Reg-binding protein

ABSTRACT

The inventors succeeded in cloning a protein binding to Reg protein from rat pancreatic Langerhans&#39; islet-derived cDNA. It was revealed that the protein expressed on the COS cell surface specifically binds to Reg protein. When the Reg-binding protein is expressed in RINm5F β cells, DNA synthesis and cell proliferation are stimulated depending on the dose of the Reg protein added to the medium, and at a higher concentration, apoptosis is induced. It is considered that the protein functions as a Reg receptor expressed on the surface of cells such as β cells and regulates the proliferation of these cells. The protein and gene thereof are useful in developing novel therapeutic agents for diabetes.

TECHNICAL FIELD

[0001] The present invention relates to a novel protein that binds tothe Reg protein, gene thereof, and production and uses of this proteinand gene.

BACKGROUND ART

[0002] β cells of pancreatic Langerhans' islet produce insulin, the soleblood hypoglycemic factor in the living body. So far, it was thoughtthat once pancreatic β cell numbers are decreased following some damage,these cells would not easily regenerate and grow. This is considered tobe an important factor in the onset of diabetes, and also the reason whya cause-based fundamental diabetes therapy cannot be established.

[0003] Conventionally, in the treatment of diabetes, insulin or an oralanti-diabetic drug of the sulfonylurea-type is administrated. However,insulin administration is a symptomatic therapy, and it is alsodifficult to maintain the physiological concentration of blood insulin.Furthermore, when considering the treatment of diabetic complicationssuch as arteriosclerosis, neuropathy, and the progression ofretinopathy, this therapy had its limitations. Moreover, prolonged useof oral anti-diabetic drugs caused side effects such as coronaryarteriosclerosis, or decrease in insulin-secreting ability thought to becaused by an excessive load to the pancreas.

[0004] The present inventors have previously demonstrated the mechanismof pancreatic β-cell damage and its prevention (H. Yamamoto, et al.,Nature 294, 284(1981); Y. Uchigata, et al., J. Biol. Chem. 257,6084(1982); Y. Uchigata, et al., Diabetes 32, 316(1983); H. Okamoto,Bioassays 2, 15(1985); H. Okamoto, J. Mol. Med. 77, 74(1999)). Further,the present inventors have succeeded in the regeneration and growth ofpancreatic β cells (T. Watanabe et al., Proc. Natl. Acad. Sci. USA 91,3589 (1994); Yonemura, Y. et al. (1984) Diabetes 33, 401-404), andisolated a gene expressing specifically during the regeneration, namedReg (Regenerating gene) (H. Okamoto, J. Mol. Med. 77, 74 (1999); K.Terazono, et al., J. Biol. Chem. 263, 2111 (1988); K. Terazono, T.Watanabe, Y. Yonemura, in Molecular biology of the islets ofLangerhans', H. Okamoto, Ed. (Cambridge University Press, Cambridge,1990), pp. 301-313; K. Terazono et al., Diabetologia 33, 250 (1990); T.Watanabe et al., Proc. Natl. Acad. Sci. USA 91, 3589 (1994)). Moreover,the present inventors elucidated that Reg protein, the gene-product ofReg gene, is a regeneration growth factor of pancreatic β cells, andshowed the possibility of treating diabetes by the administration of theReg protein, the activation of Reg gene, or the introduction of Reggene, by using a diabetes model animal (Watanabe, T. et al. (1994) Proc.Natl. Acad. Sci. USA 91, 3589-3592; Gross, D. J. et al. (1998)Endocrinology 139, 2369-2374; Okamoto, H. (1999) J. Mol. Med. 77,74-79). From these analysis, administration of Reg protein was found toinduce the regeneration and growth of β-cells, thereby increasing β-cellmass and amelioration of diabetes in 90% of pancreatectomized rats andin non-obese diabetic mice. However, it was unknown as to which proteinsinteract with the Reg protein to exert its functions.

[0005] Reg protein is expected to be applied to diabetes treatment as agrowth factor of pancreatic β cells, to make up for the weak-points ofinsulin administration. However, a lot of technical issues still existwhen it comes to clinical application, such as that oral administrationof Reg protein is difficult due to its high-molecular weight, andfurthermore, the in vivo targeting of a high-molecular weight protein isdifficult.

DISCLOSURE OF THE INVENTION

[0006] An objective of the present invention is to provide a novelprotein binding to Reg protein, gene thereof, and methods of productionand uses of the protein and gene. Especially, the protein of the presentinvention is useful for the development of a novel therapeutic drug fordiabetes.

[0007] In order to analyze the function of Reg protein towardspancreatic β cell-lineage cells, the present inventors conducted anexperiment in which a recombinant Reg protein produced in yeast wasadded to the rat insulinoma cell-derived cell line, RINm5F. As a result,it was revealed that the addition of Reg protein increases incorporationof 5′-bromo-2′-deoxyuridine (BrdU) in RINm5F cells significantly, andthat the growth of these cells is promoted by Reg protein. Next, thepresent inventors labeled the Reg protein with ¹²⁵I and added it toRINm5F cells to analyze the binding activity. As a result,concentration-dependent binding of Reg protein to RINm5F cells wasobserved, and the binding was thought to be specific since it wasinhibited by an excess amount of unlabeled Reg protein. These resultssuggest that pancreatic β cells express a Reg protein receptor and thebinding of this receptor to Reg protein promotes cell growth.

[0008] To isolate a Reg-binding protein that functions as a Reg proteinreceptor, the present inventors constructed an expression cDNA libraryfrom rat pancreatic Langerhans' islet polyA (+) RNA by a phage vectorand screened genes encoding a Reg-binding protein by West-Westernblotting method using a labeled Reg protein. As a result, a novel cDNAencoding a protein comprising 364 amino acids was successfully isolated.This cDNA was inserted into a mammalian cell-expression vector, andexpressed in COS-7 cells. Addition of recombinant Reg protein to thesecells confirmed that Reg protein bound specifically to COS-7 cells.

[0009] Using this cDNA as a probe, the present inventors succeeded inisolating another cDNA encoding a Reg-binding protein by screening a ratpancreas Langerhans' islet cDNA library. The cDNA was encoding a cellsurface protein comprising 919 amino acids. When the cDNA was expressedin mammalian cells, the protein was expressed on the cell surface andthe cells bound to Reg protein with a high affinity. The addition of Regprotein induced the incorporation of BrdU in RINm5F β cells transfectedwith the cDNA, and the cell number was increased. From these results, itwas shown that the Reg-binding protein encoded by the isolated cDNA wasa receptor for Reg protein, and mediated cell proliferation signals inpancreatic β cells. Moreover, it was revealed that apoptosis is inducedin RINm5F cells highly expressing the Reg-binding protein by theaddition of a high concentration of Reg protein.

[0010] From these facts, it can be envisaged that the Reg-bindingprotein transduces signals of Reg protein, and by regulating cellproliferation, and such, of pancreatic β cells, the Reg-binding proteinregulates pancreatic β cell mass. The Reg-binding protein of the presentinvention and gene thereof would be useful tools for elucidating theetiological mechanism of diabetes, and these can also be applied to thedevelopment of anti-diabetic drugs.

[0011] The present invention relates to a Reg protein-binding protein,gene thereof, and methods for producing the protein and gene, and usesthereof, more specifically to:

[0012] (1) a DNA according to any one of (a) to (i),

[0013] (a) a DNA encoding a protein comprising the amino acid sequenceof SEQ ID NO: 2,

[0014] (b) a DNA comprising the coding sequence of the nucleotidesequence of SEQ ID NO: 1,

[0015] (c) a DNA encoding a protein comprising an amino acid sequence inwhich one or more amino acids of the amino acid sequence of SEQ ID NO: 2have been substituted, deleted, inserted and/or added, wherein said DNAencodes a protein having the activity of binding to Reg protein,

[0016] (d) a DNA hybridizing to a DNA comprising the nucleotide sequenceof SEQ ID NO: 1, wherein said DNA encodes a protein having the activityof binding to Reg protein,

[0017] (e) a DNA encoding a protein comprising the amino acid sequenceof SEQ ID NO: 4,

[0018] (f) a DNA comprising the coding region of the nucleotide sequenceof SEQ ID NO: 3,

[0019] (g) a DNA encoding a protein comprising the amino acid sequencein which one or more amino acids of the amino acid sequence of SEQ IDNO: 4 have been substituted, deleted, inserted and/or added, wherein theDNA encodes a protein having the activity of binding to Reg protein,

[0020] (h) a DNA hybridizing to a DNA comprising the nucleotide sequenceof SEQ ID NO: 3, wherein said DNA encodes a protein having the activityof binding to Reg protein,

[0021] (i) a DNA encoding a partial peptide of a protein comprising theamino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4;

[0022] (2) a protein or peptide encoded by the DNA according to (1);

[0023] (3) a vector into which the DNA according to (1) has beeninserted;

[0024] (4) a host cell carrying the vector according to (3);

[0025] (5) a method for producing the protein or peptide according to(2) wherein said method comprises the following steps of,

[0026] (a) culturing the cell according to (4), and,

[0027] (b) recovering the recombinant protein expressed by the cell fromthe cultured cell or from the culture supernatant;

[0028] (6) an antibody against the protein or peptide according to (2);

[0029] (7) a polynucleotide comprising at least 15 nucleotides, whereinsaid polynucleotide hybridizes with a DNA selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, and DNA complementary thereto;

[0030] (8) a method of screening for a compound that binds to theprotein or peptide according to (2), wherein said method comprises thefollowing steps of,

[0031] (a) contacting the protein or peptide with a test sample,

[0032] (b) detecting the binding of the test sample to the protein orpeptide, and,

[0033] (c) selecting a compound that binds to the protein or peptide;

[0034] (9) a method of screening for a compound that inhibits thebinding of Reg protein to the protein or peptide according to (2),wherein said method comprises the following steps of,

[0035] (a) contacting Reg protein with the protein or peptide accordingto (2) in the presence of a test sample,

[0036] (b) detecting the binding of Reg protein to the protein orpeptide according to (2), and,

[0037] (c) selecting a compound that decreases the binding;

[0038] (10) a compound isolated by the method according to (9), whereinsaid compound inhibits the binding of Reg protein to the protein orpeptide according to (2);

[0039] (11) a method of screening for a compound that promotes orinhibits signal transduction caused by an activation of the proteinaccording to (2), wherein said method comprises the following steps of,

[0040] (a) contacting Reg protein with a cell expressing the proteinaccording to (2) on the cell surface, in the presence of a test sample,

[0041] (b) detecting a change of the cell in response to the stimulationby Reg protein,

[0042] (c) selecting a compound that enhances or suppresses the changeof the cell as compared to when detected in the absence of the testsample;

[0043] (12) the method according to (11), wherein said change of thecell detected comprises a change in cell-proliferating activity orDNA-synthesizing activity of the cell;

[0044] (13) a compound isolated by the method according to (11) or (12),wherein said compound promotes or inhibits signal transduction caused byan activation of the protein according to (2);

[0045] (14) a pharmaceutical agent comprising the DNA according to (1),the protein or peptide according to (2), the vector according to (3),the antibody according to (6), or the compound according to (10) or(13);

[0046] (15) the pharmaceutical agent according to (14), wherein saidpharmaceutical agent is selected from the group consisting of aReg-binding agent, a regulator of intracellular signal transduction ofcells responding to Reg protein, a cell growth regulator, a DNAsynthesis regulator, and an apoptosis regulator; and,

[0047] (16) the pharmaceutical agent according to (14) or (15), whereinsaid pharmaceutical agent is an anti-diabetic drug.

[0048] The present invention relates to a novel protein expressed in thepancreas that binds to the Reg protein (Reg-binding protein). Thenucleotide sequences of cDNAs of isolated rat “Reg-binding protein” andamino acid sequences encoded by these cDNA are described in SEQ ID NO: 1and SEQ ID NO: 3, and SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0049] One of the cDNA encoding the rat Reg-binding protein of thepresent invention (SEQ ID NO: 2) comprises an open reading frameencoding a protein comprising 364 amino acid residues (SEQ ID NO: 1). Bya screening using this cDNA as probe, a cDNA encoding Reg-bindingprotein comprising an open reading frame (SEQ ID NO: 3) encoding aprotein comprising 919 amino acid residues (SEQ ID NO: 4) could beisolated. The rat “Reg-binding protein” of the present invention isexpressed on the cell surface and has a Reg protein-binding activity. Asdescribed above, the Reg protein is a regeneration growth factor that isspecifically expressed when pancreatic β cells are regenerated, and thepossibility of applying this protein and the gene thereof in treatingdiabetes has been suggested. It is thought that the Reg-binding proteinof the present invention relates to the regulation of physiologicalfunctions of cells including growth regulation of pancreatic β cells, byfunctioning as a receptor of the Reg protein. Therefore, the Reg-bindingprotein of the present invention maybe useful as a research target forelucidating the mechanism that causes diabetes, or as a tool fordeveloping a therapeutic agent against diseases involving pancreatic βcell functions (such as diabetes).

[0050] Recently, several Reg and Reg-related genes have been isolated,and these have been revealed to constitute a multigene family, the Regfamily (H. Okamoto, J. Mol. Med. 77, 74 (1999); H. Okamoto, J.Hepatobiliary Pancreat. Surg. 6, 254 (1999); M. Unno et al., J. Biol.Chem. 268, 15974 (1993); Y. Narushima et al., Gene 185, 159 (1997); M.Abe et al., Gene 246, 111 (2000)). All the members of the Reg familyshow the conserved gene organization of 6 exons and 5 introns and 40-85%amino acid sequence homologies among the family with the conserved 6cysteine residues forming 3 pairs of intramolecular S—S bonds (H.Okamoto, J. Mol. Med. 77, 74 (1999); H. Okamoto, J. HepatobiliaryPancreat. Surg. 6, 254 (1999); M. Unno et al., J. Biol. Chem. 268, 15974(1993); Y. Narushima et al., Gene 185, 159 (1997); T. Itoh et al., FEBSLett. 272, 85 (1990); M. Abe et al., Gene 246, 111 (2000)). Based on theprimary structures of Reg proteins, the members of the family aregrouped into three subclasses, type I, II and III (H. Okamoto, J. Mol.Med. 77, 74 (1999); H. Okamoto, J. Hepatobiliary Pancreat. Surg. 6, 254(1999); M. Unno et al., J. Biol. Chem. 268, 15974 (1993); Y. Narushimaet al., Gene 185, 159 (1997); T. Watanabe et al., J. Biol. Chem. 265,7432 (1990); M. Abe et al., Gene 246, 111 (2000)). Type I Reg proteins,which include the rat and human Reg proteins used in the examples of thepresent invention, are expressed in regenerating pancreatic islets (H.Okamoto, J. Mol. Med. 77, 74 (1999); K. Terazono, et al., J. Biol. Chem.263, 2111 (1988); K. Terazono, T. Watanabe, Y. Yonemura, in Molecularbiology of the islets of Langerhans', H. Okamoto, Ed. (CambridgeUniversity Press, Cambridge, 1990), pp. 301-313; K. Terazonoetal.,Diabetologia 33, 250 (1990); H. Okamoto, J. Hepatobiliary Pancreat.Surg. 6, 254 (1999)). Recently, type I Reg expression under pathologicalconditions has been reported in human colon cancer (T. Watanabe et al.,J. Biol. Chem. 265, 7432 (1990); M. E. Zenilman et al., J. Gastrointest.Surg. 1, 194 (1997); F. R. Bernard-Perrone et al., J. Histochem.Cytochem. 47, 863 (1999)), and in rat gastric mucosa (H. Fukui et al.,Gastroenterology 115, 1483 (1998)) and enterochromaffin-like cells (M.Asahara et al., Gastroenterology 111, 45-(1996)), and type III Regproteins have also been suggested to be involved in cellularproliferation in intestinal Paneth cells (L. Christa et al., Am. J.Physiol. 271, G993 (1996)), hepatocellular carcinomas (L. Christa etal., Am. J. Physiol. 271, G993 (1996)), pancreatic acinar cells (L.Christa et al., Am. J. Physiol. 271, G993 (1996); E. M. Ortiz et al.,Gastroenterology 114, 808 (1998)) and Schwann cells (J. F. Livesey etal., Nature 390, 614 (1997)). Therefore, the identified Reg receptor mayfunction in various tissues and cells in physiological and pathologicalconditions as a receptor for the Reg family gene products.

[0051] As shown by findings described above, the protein of the presentinvention is useful for the development of a therapeutic agent for thetreatment and prevention of not only diabetes, but also diseases such asgastrointestinal tumors (Asahara, M. et al., Gastroenterology 111, 45-55(1996); Fukui, H. et al., Gastroenterology 115, 1483-1493 (1998)),neurodegeneration diseases (Livesy, F. J. et al., Nature 390, 614-618(1997)), and pancreatitis (Christa, L. et al., Am. J. Phsiol. 271,G993-G1002 (1996); Ortiz, E. et al., Gastroenterology 114, 808-816(1998)). Moreover, it is thought that Reg protein itself can be appliedfor the treatment when Reg protein-Reg-binding protein disorders, forexample, overstimulation, occur in tumors and such, since theadministration of the soluble form of Reg-binding protein can inhibitthe overstimulation to suppress tumor growth, etc.

[0052] The present invention includes proteins structurally similar torat “Reg-binding protein”, as long as they have a binding activity toReg protein. Structurally similar proteins include mutants of“Reg-binding protein” and “Reg-binding proteins” derived from otherorganisms.

[0053] One skilled in the art could readily prepare these proteinsusing, for example, well-known mutagenesis methods. Known methods foraltering amino acids in proteins include Kunkel's method (Kunkel, T. A.(1985) Proc. Natl. Acad. Sci. USA 82, 488), Oligonucleotide-directedDual Amber (ODA) method (Hashimoto-Gotoh, T. et al. (1995) Gene 152,271-275), PCR-restriction enzyme method (Ito, W. et al. (1991) Gene 102,67-70), ODA-PCR method (Hashimoto-Gotoh, T. et al. (1995) Gene 152,271-275; Ito, W. et al. (1991) Gene 102, 67-70), etc. There is norestriction on the number of amino acid residues altered, but whenartificially doing so, the number of amino acid residues altered isusually 50 or less, preferably 10 or less, and more preferably 5 orless.

[0054] Mutation of amino acids in proteins could occur spontaneously.Such proteins having amino acid sequences different from that of thenatural rat “Reg-binding protein” due to artificial or spontaneoussubstitution, deletion, addition and/or insertion of amino acids, arealso included in this invention as long as they have a binding activityto Reg protein.

[0055] An amino acid having properties similar to those of thesubstituted amino acid is preferably used for the substitution. Forexample, since Ala, Val, Leu, Ile, Pro, Met, Phe and Trp are, classifiedas non-polar amino acids, they are considered to have similarproperties. Moreover, non-charged amino acids include Gly, Ser, Thr,Cys, Tyr, Asn, and Gln. Furthermore, acidic amino acids include Asp andGlu, while basic amino acids include Lys, Arg and His.

[0056] In the present invention, a protein that is deficient in aminoacids of rat “Reg-binding protein” includes a protein comprising onlythe extracellular domain. Moreover, a protein comprising an amino acidaddition to rat “Reg-binding protein” includes a fusion protein of rat“Reg-binding protein” and another peptide.

[0057] Proteins structurally similar to the rat “Reg-binding protein”having a binding activity towards Reg protein can be prepared using aknown hybridization technique (Sambrook, J. et al. (1989) MolecularCloning 2nd ed., Cold Spring Harbor Laboratory Press) and polymerasechain reaction (PCR) technique (Sambrook, J. et al. (1989) MolecularCloning 2nd ed., Cold Spring Harbor Laboratory Press; Innis, M. A. etal., PCR Protocols, Academic Press (1990)). Namely, it is routine forone skilled in the art to isolate a DNA highly homologous to rat“Reg-binding protein” cDNA from various other organisms using the rat“Reg-binding protein” cDNA (SEQ ID NO: 1 or 3), or portions thereof, asprobe, and oligonucleotides specifically hybridizing to the rat“Reg-binding protein” cDNA as primer, to obtain proteins structurallysimilar to the rat “Reg-binding protein” from the isolated DNA.

[0058] A protein encoded by DNA hybridizing to the rat “Reg-bindingprotein” cDNA is included in this invention, as long as it has a bindingactivity towards rat “Reg-binding protein”. Other organisms used forisolating such a protein include, for example, humans, monkeys, mice,rabbits, goats, cattle, pigs, dogs and so on, but are not limitedthereto. β cells of pancreatic Langerhans' islet of these organisms arethought to be a suitable source when isolating DNA encoding such aprotein.

[0059] DNAs encoding the “Reg-binding protein” derived from organismsother than rats are usually highly homologous to the cDNA sequence (SEQID NO: 1 or 3) of rat “Reg-binding protein”. “Highly homologous” meansat least 60% or more, preferably 80% or more, and more preferably 90% ormore, even more preferably 95% or more, most preferably 99% or moresequence identity at the nucleotide sequence level. The homology of thesequence can be determined by FASTA (searches one with wide rangesequence similarity), BLAST (searches one with locally high similarity)and SSEARCH (search employing Smith-Waterman algorithm). These can beused by going to well-known databases and websites such as DNA Data Bankof Japan (DDBJ).

[0060] Hybridization conditions for isolating, from an organism otherthan the rat, a cDNA encoding a protein functionally equivalent to rat“Reg-binding protein” using rat “Reg-binding protein” cDNA, can besuitably selected by one skilled in the art. For example, hybridizationcan be carried out at 42° C. using 6×SSC, 5×FBP, 0.5% SDS, 0.2 mg/mlsalmon (herring) sperm DNA, and 10% formamide solution (low-stringentconditions). Preferably, the hybridization is carried out at 42° C.using 6×SSC, 5×FBP, 0.5% SDS, 0.2 mg/ml salmon (herring) sperm DNA, and30% formamide solution (medium-stringent conditions). More preferably,the hybridization is carried out at 50° C. using 6×SSC, 5×FBP, 0.5% SDS,0.2 mg/ml salmon (herring) sperm DNA, and 50% formamide solution(highly-stringent conditions). In this case, although several factorsincluding temperature, formamide concentration, salt concentration, andsuch are thought to influence the stringency of hybridization, oneskilled in the art can accomplish similar stringencies by suitablyselecting these factors.

[0061] The protein of this invention can be prepared as either a naturalprotein or a recombinant protein utilizing gene recombinationtechniques. A natural protein can be prepared by, for example,subjecting extracts from tissues that are thought to express the“Reg-binding protein” (for example, β cells of pancreatic Langerhans'islet) to affinity chromatography using an antibody against the“Reg-binding protein” as described below. On the other hand, arecombinant protein can be prepared by culturing cells transformed withDNA encoding the “Reg-binding protein”, allowing the transformants toexpress the protein, and recovering the protein as described below.

[0062] The present invention includes partial peptides of the protein ofthe present invention. An example of partial peptides of the proteins ofthe present invention is a peptide corresponding to the Regprotein-binding site. By administering a partial peptide of the presentinvention to a living body, it can be utilized as an agonist orantagonist of the protein of the present invention, or an antagonist,and such, of the Reg protein. Such partial peptides are useful asactivators or inhibitors of signal transduction mediated by the proteinof this invention. Additionally, the partial peptides of this inventioninclude a partial peptide of the N-terminal region, or the C-terminalregion of the protein of this invention, and these peptides can beutilized to prepare antibodies. Partial polypeptides comprising aminoacid sequences specific to the protein of this invention have at least7, preferably at least 8, more preferably at least 9 amino acidresidues. Partial peptides of this invention can be produced by, forexample, genetic engineering techniques, known peptide synthesizingmethods, or by cleaving the protein of this invention with appropriatepeptidases. For example, partial peptides comprising domains binding toReg protein can be used for binding to Reg protein. Such partialpeptides can be used as Reg protein-binding agents.

[0063] This invention relates to DNAs encoding the protein of theinvention. DNA encoding the protein of this invention is notparticularly limited as long as it can encode the protein of thisinvention, and includes cDNA, genomic DNA, and synthetic DNA. DNA havingany nucleotide sequence based on the degeneracy of genetic codes is alsoincluded in this invention as long as they can encode the protein ofthis invention.

[0064] cDNA encoding the protein of this invention can be screened, forexample, by labeling cDNA of SEQ ID NO: 1 or 3 or fragments thereof, RNAcomplementary to them, or synthetic oligonucleotides comprising partialsequences of the cDNA with ³²P and such, and hybridizing them to a cDNAlibrary derived from tissues (e.g., pancreas, etc.) expressing theprotein of this invention. Also, such cDNAs can be cloned bysynthesizing oligonucleotides corresponding to nucleotide sequences ofthe cDNAs, and amplifying them by polymerase chain reaction with cDNAderived from suitable tissues (e.g. pancreas, etc.) as a template.Genomic DNA can be screened, for example, by labeling cDNA of SEQ ID NO:1 or 3 or segments thereof, RNA complementary to them, or syntheticoligonucleotides comprising partial sequences of the cDNA with ³²P andsuch, and hybridizing them with a genomic DNA library. Alternatively,the genomic DNA can be cloned by synthesizing oligonucleotidescorresponding to nucleotide sequences of these cDNAs, and amplifyingthem by polymerase chain reaction using genomic DNA as a template. Onthe other hand, synthetic DNAs can be prepared, for example, bychemically synthesizing oligonucleotides comprising partial sequences ofcDNA of SEQ ID NO: 1 or 3, annealing them to form a double strand, andligating them by DNA ligase.

[0065] These DNAs are useful for the production of recombinant proteins.Namely, the protein of the present invention can be prepared as arecombinant protein by inserting DNAs encoding the protein of thisinvention (e.g. SEQ ID NO: 1 or 3) into an appropriate expressionvector, transforming suitable cells with the vector, culturing thetransformants, and recovering the expressed protein. The protein of thepresent invention can be prepared as a purified or crude protein, or inthe membrane-bound form after expressing in mammalian cells.

[0066] Example of specific host-vector systems are, E. coli-pGEX system(Amersham Pharmacia Biotech; expressed as GST-fusion protein), E.coli-pHB6 system and pVB6 system (Roche diagnostics; expressed as6×His-fusion protein), E. coli-pMAL system (New England Biolabs;expressed as a fusion protein with maltose-binding protein), E.coli-pTYB system (New England Biolabs; expressed as a fusion proteinwith Intein (Intein part is digested under the presence of DTTfacilitating purification of only the objective protein), Pichia-pPICsystem and pGAP system (Invitrogen), mammalian cells (for example,COS7)-pCI-neo system (Promega) and pHook system (Invitrogen), and such.

[0067] Vectors can be introduced into hosts by the well knowntransformation into competent cells or electroporation for E. coli,transformation into competent cells prepared with Pichia Easy Comp kit(refer to Example 1) or electroporation for Pichia, electroporation orwell known lipofection method using cationic lipids for mammalian cells,etc.

[0068] Recombinant proteins expressed in host cells can be purified byknown methods. The protein of this invention expressed in the form of afusion protein, for example, with a histidine residue tag orglutathione-S-transferase (GST) attached at the N-terminus can bepurified by a nickel column or a glutathione sepharose column, etc.

[0069] DNA encoding the protein of the present invention can also beapplied to gene therapy against diseases caused by a mutation therein.For example, gene therapy using a vector of a virus such as the vacciniavirus or retrovirus can be given. An actual therapeutic method would be:introducing “Reg-binding protein” into, for example, pancreas or theLangerhans' islets to be used in a transplantation, under cultureconditions using these recombinant viruses, and conductingtransplantation. This would improve the therapeutic effects of thetransplantation through the proliferation of pancreatic β cells, andenable effective use of the transplanting organ.

[0070] The present invention also relates to a polynucleotide comprisingat least 15 nucleotides hybridizing to DNA comprising the nucleotidesequence described in SEQ ID NO: 1 or SEQ ID NO: 3, or complimentary DNAthereof. The polynucleotide preferably hybridizes specifically to DNAcomprising the nucleotide sequence described in SEQ ID NO: 1 or SEQ IDNO: 3, and comprises at least 15 nucleotides. “Hybridize specifically”means that no significant cross-hybridization with DNA encoding otherproteins is observed under the normal hybridization conditions,preferably under the medium-stringent hybridization conditions describedabove, more preferably under the highly stringent hybridizationconditions described above. Hybridization can be conducted at theconditions described above. These polynucleotides include probes andprimers, nucleotides or nucleotide derivatives (for example, antisenseoligonucleotides and ribozymes), which can specifically hybridize to DNAencoding the protein of the present invention or the DNA complementaryto the DNA.

[0071] Oligonucleotides comprising cDNA encoding the protein of theinvention or a partial sequence thereof can be used for the cloning ofgenes or cDNA encoding the protein of the present invention or theamplification by PCR. Moreover, they are useful for the detection andquantification of RNA encoding the protein of the present invention.Furthermore, they can be used for detecting a mutation, polymorphism, ordisorder (such as gene diagnosis), by methods such as restrictionfragment length polymorphism (RFLP), single strand conformationpolymorphism (SSCP).

[0072] The polynucleotide of the present invention can be used forpancreatic tests, for example a pancreatic β cell test, since theprotein of the present invention has important functions in theformation, regeneration and/or maintenance of the pancreas, especiallyin the regulation of pancreatic β cell mass. Moreover, thepolynucleotide of the present invention can be used in diabetes tests.For example, pancreatic tissue samples are isolated from subjects andabnormalities in the expression levels of the protein of the presentinvention in these tissues can be examined by methods such as northernhybridization, RT-PCR, or DNA chip (DNA microarray) Moreover, thepresence or absence of a mutation or polymorphism of the DNA or RNAencoding the protein of the present invention can be tested by sequenceanalysis, SSCP, RFLP, etc. In the case of using the polynucleotide as atest reagent, it can be properly mixed with distilled water, a buffer,salt, and so on.

[0073] Moreover, the protein of the present invention or partialpeptides thereof, DNA encoding the protein or peptides, and vectors intowhich the DNA has been inserted can be used for the below-mentionedscreening of compounds inhibiting the binding of the protein of thepresent invention and Reg protein. It can also be used for screeningcompounds promoting or inhibiting the signal transduction (for example,cell growth activity or DNA-synthesizing activity of cells) stimulatedby the activation of the protein of the present invention. Thesescreenings can be applied for assaying therapeutic agents or preventivedrugs for diseases caused by disorders in the mass or functions ofpancreatic β cells, including diabetes. The screenings can also be usedfor assaying or screening therapeutic agents or preventive drugs forgastrointestinal tumors, neurodegeneration diseases, pancreatitis, andother tumors, besides diabetes.

[0074] Moreover, the present invention relates to an antibody binding tothe protein of the present invention. The antibody of the presentinvention includes polyclonal and monoclonal antibodies. A polyclonalantibody can be prepared by immunizing a rabbit, goat, sheep, or such bya well known method (Harlow, E. and Lane, D. Antibodies, Cold SpringHarbor Laboratory (1988), etc.) using as the antigen a “Reg-bindingprotein” prepared from a biomaterial (for example, pancreas Langerhans'islet), a recombinant “Reg-binding protein” produced by a host-vectorsystem, and such described above, or partial peptides synthesized byordinary peptide synthesis methods. A monoclonal antibody can beprepared by immunizing a mouse, rat, or such, by a well known method(Harlow, E. and Lane, D. Antibodies, Cold Spring Harbor Laboratory(1988), etc.) using as the antigen a “Reg-binding protein” prepared froma biomaterial (for example, pancreas Langerhans' islet), a recombinant“Reg-binding protein” produced by a host-vector system, and suchdescribed above, or partial peptides synthesized by ordinary peptidesynthesis methods, and using splenocytes of the mouse, rat, or such, toobtain a hybridoma which produces the monoclonal antibody.

[0075] Antibodies are purified by ordinary biochemical methods such asammonium sulfate fractionation, protein G Sepharose column, or affinitycolumns in which an antigen is immobilized, from serum in the case ofpolyclonal antibodies, and from the culture supernatant of hybridoma orascites of animals inoculated with the hybridoma in the case ofmonoclonal antibodies.

[0076] Antibodies thus prepared are used for the affinity purificationof the proteins of this invention or, can be used for testing anddiagnosing disorders caused by abnormal expression or structuralabnormalities of the protein of this invention and for detecting theexpression level of the protein, etc. Specifically, for example,proteins are extracted from tissues or cells, and through the detectionof protein of the present invention by Western blotting,immunoprecipitation, ELISA, and such, abnormalities in the expression orstructure can be tested and/or diagnosed. The antibody of the presentinvention can be also used for pancreatic tests, for example, pancreaticβ cell tests. Moreover, the antibody of the present invention can beused for testing diabetes. For example, by isolating a pancreatic tissuesample from a subject, abnormalities in the expression level orstructure of the protein of the present invention in the tissue can betested by Western blotting, immunohistochemistry, ELISA, EIA, and such.In the case of using the antibody as a test reagent, sterilized water,buffer, salt, stabilizer, preservative, and such can be combinedappropriately. Moreover, the antibody of the present invention may alsobe used for antibody therapy. In the case of using the antibody of thepresent invention for antibody therapy, humanized or human antibodiesare preferable. In this case, human lymphocytes and HGPRT(hypoxanthine-guanine phosphoribosyl transferase)-deficient myelomacells are fused and human-mouse heterohybridomas are selected using HATmedium. Myeloma cells are selected by the well-known RIA or ELISA methodin which “Reg-binding protein” is used as the antigen, and clonesproducing humanized monoclonal antibody are obtained. Purification ofthe antibody can be conducted as described above.

[0077] The present invention also relates to a method for screening acompound binding to the protein of this invention. Such a screening canbe carried out by a method comprising the following steps: (a)contacting the protein of the invention or its peptide with a testsample, (b) detecting the binding of the test sample to the protein ofthe invention or its peptide, and, (c) selecting a compound that bindsto the protein of the invention or its peptide.

[0078] The protein of the present invention can be used for thescreening as a purified protein, in the cell surface-expressed form, oras a cell membrane fraction, according to the method of screening.

[0079] Test samples, for example, cell extracts, expression products ofgene libraries, synthetic low molecular weight compounds, syntheticpeptides, natural compounds, and such, can be used, but are not limitedthereto. The test samples used for screening can be labeled prior to useas necessary. Labels include, for example, radioactive and fluorescentlabels, and such, but are not limited thereto.

[0080] Screening of a protein binding to the protein of the presentinvention can be carried out, for example, by applying the culturesupernatant of cells, or cell extract expected to express proteinsbinding to the protein of this invention, to an affinity column in whichthe protein of this invention has been immobilized, and by purifying aprotein that specifically binds to this column.

[0081] Moreover, it can be conducted according to “West-Western blottingmethod”, and such, in which a cDNA library is constructed from tissuesor cells (for example, pancreatic β cells) expected to express theprotein binding to the protein of the present invention, and then, thisis expressed on agarose and the protein expressed is immobilized on thefilter and reacted with labeled protein of the present invention todetect plaques expressing the binding protein. Another method is the“two-hybrid system” in which GAL4-DNA binding domain and GAL4transcriptional activation domain are expressed as a fusion protein ofthe present invention and the test protein, and the binding of theprotein of the present invention and the test protein is detectedthrough the expression of a reporter gene linked to the downstream of apromoter with the binding sequence of GAL4-DNA binding protein.

[0082] Moreover, the method in which the immobilized protein of thepresent invention is reacted with a synthetic compound, natural productbank, or a random phage peptide display library to screen the bindingprotein, and the method in which a compound binding to the protein ofthe present invention is isolated by screening by combinatorialchemistry techniques using high-throughput system, are techniques wellknown to one skilled in the art.

[0083] Moreover, a screening using BIACORE (Biacore), or a method inwhich changes in acid secretion speed of cultured cells forced toexpress Reg-binding protein of the present invention are monitored byusing a microphysiometer (Molecular Device), and such, can be given asexamples.

[0084] Moreover, the present invention relates to a method for screeninga compound that inhibits the binding of the protein of the presentinvention and Reg protein. Such a screening can be conducted by a methodincluding the following steps of: (a) contacting Reg protein with theprotein of the present invention in the presence of a test sample, (b)detecting the binding of Reg protein to the protein of the presentinvention, and, (c) selecting a compound that decreases the binding.

[0085] The protein of the present invention can be used for thescreening as a purified protein, in the cell surface-expressed form, oras a cell membrane fraction. Reg protein is usually used for screeningas a purified protein. For example, human REG Iα or rat Reg I, and suchcan be used as the Reg protein. These proteins can be prepared asrecombinant proteins (refer to Example 1). Reg protein can be labeledwith radioisotopes such as [¹²⁵I], if necessary.

[0086] As test samples, for example, cell extract solutions, expressionproducts of gene libraries, synthesized low molecular compounds,synthesized peptides, natural compounds, and such can be used, but arenot limited thereto.

[0087] Screening can be conducted, for example, as follows. Cellsexpressing the protein of the present invention or a membrane fractionprepared using them are contacted with a labeled ligand (Reg protein)under the presence of a test sample, and the amount of the labeledligand binding to the protein of the present invention is measured. Acompound that lowers the amount of ligand as compared with the case inthe absence of the test sample is selected. The binding of the proteinof the present invention and Reg protein can be measured using BIACOREor microphysiometer described above. Compounds thus isolated can be thecandidates for antagonists or agonists of the protein of the presentinvention.

[0088] Moreover, the present invention relates to a method for screeninga compound that promotes or inhibits the signal transduction caused bythe activation of the protein of the present invention. Such a screeningcan be conducted by the following steps: (a) contacting Reg protein witha cell expressing the protein of the present invention on the cellsurface in the presence of a test sample, (b) detecting a change of thecell in response to the stimulation by Reg protein, (c) selecting acompound that enhances or suppresses the change of the cell as comparedto when detected in the absence of the test sample (control).

[0089] Cells expressing the protein of the present invention on itssurface can be prepared by inserting DNA encoding the protein of thepresent invention into an appropriate expression vector and introducingit to appropriate host cells. For example, cells such as RINm5F cells,CHO cells, COS-7 cells can be given as host cells. As for the vector,pCI-neo (Promega), pHook (Invitrogen) and such can be given.

[0090] As test samples, for example, cell extracts, expression productsof the gene libraries, synthesized low molecular compounds, synthesizedpeptides, natural compounds and such are used, but are not limitedthereto. Moreover, as a test sample, it is also possible to use acompound isolated by the above-described screenings using as an indexthe binding with the protein of the present invention.

[0091] Reg protein is used for screening usually as a purified protein.As the Reg protein, for example, human REG Iα or rat Reg I, and such canbe used. These proteins can be prepared as recombinant proteins (referto Example 1).

[0092] In the screening, a change of the cells described above inresponse to the Reg protein stimulation under the presence of a testsample is detected. As the change of the cells in response to the Regprotein stimulation, for example, a change in cell growth activity, achange in DNA synthesis activity, a change in the degree of apoptosis ofcells, phosphorylation of the protein of the present invention orproteins transducing signals, a change in the expression of a specificgene in the cells, and such, can be given, but the change is not limitedthereto.

[0093] The DNA synthesis of cells can be detected, for example, asindicated in examples, by measuring the incorporation of5′-bromo-2′-deoxyuridine (BrdU). Moreover, the detection can beconducted by measuring radioactivity, incorporated after the addition of³H-thymidine to cells. The test of ³H-thymidine incorporation to cellsis generally used to assay the promotion or inhibition effect on DNAsynthesis. The method has the advantages of enabling the handling of arelatively large amount of samples, with a high sensitivity, etc. In thescreening of a compound promoting or inhibiting DNA synthesis,specifically, for example, cells are seeded onto a multi-well plate andsuch, and after 1-2 day incubation, medium is changed to a mediumcontaining the test sample and incubated for certain duration such as 24hours. Thereafter, for example, 1 μCi/ml of ³H thymidine is added. Afterincubating, the medium is removed, washed, 10% TCA is added, and then,the cells are left to stand for approximately 20 min, and washed withice cold 5% TCA. The cells are then lysed with 0.5 N NaOH, left to standon ice for 10 min, ½ volume of 1 N HCl is added and gently mixed, then,40% TCA is added to a final concentration of 10%, and gently mixed.After standing on ice for 20 min, the solution is filtrated by a WhatmanGF/C filter, and such, to collect insoluble material. After washing with100% ethanol for 3 times and drying, radioactivity is measured using aliquid scintillation counter.

[0094] Moreover, the cell growth can be measured by measuring cellnumbers or colony numbers, or by measuring a color development that isdependent on the cell number by adding dyes such as MTT or Alamar Blue.The MTT method measures cell growth activity using color development byMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), andMTT formazan is formed due to a reaction with the respiratory chain ofmitochondria of living cells. The amount produced reflects the cellnumber. Specifically, for example, cells are incubated in a 96-wellplate, reacted with a test sample, and then, 10 μl of 5 mg/ml MTTsolution is added, and incubated for 4 hours. Then, 100 μl of 0.04 NHCl/isopropanol is added, mixed well, and left to stand for severalminutes. Then, the coloring is measured using a microplate reader at thereference wavelength of 630 nm and test wavelength of 570 nm. Moreover,as described in Example 11, tetrazolium salt 4[-3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolyo]-1,3-benzenedisulfonate(WST-1) can be used for the assay.

[0095] The apoptosis of cells can be assayed, for example, usingmorphological changes in the nucleus (condensation or segmentation ofnucleus), fragmentation of chromosomes (ladder formation) and such, asindexes. Specifically, apoptosis can be detected, for example, by theTUNEL method (Y. Gavrieli et al., J. Cell Biol. 119, 493(1992)), and soon (refer to Example 11).

[0096] Protein phosphorylation is considered to occur in serine,threonine or tyrosine residues. These changes of phosphorylation can bedetected by measuring the phosphorylation state of intracellularproteins by Western blotting method or immunoprecipitation method usinganti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosineantibodies. Cell proliferation-related proteins such as MAP kinasefamily, STAT family, or Fos-Jun family protein can be expected to bephosphorylated, but are not limited thereto.

[0097] It is known that transcription of various genes is induced orsuppressed by the protein phosphorylation described above, etc. Changesin expression of a specific gene depending on the binding of the proteinof the present invention and its ligand can be detected using a reportergene. Namely, the change in expression can be measured by detectingreporter gene expression in which the reporter gene is linked to thedownstream of the promoter of the gene. Moreover, a change in expressionof a specific gene can also be measured by northern blotting or RT-PCRmethod in which mRNA is detected, a method using an antibody to detectproteins that are gene translation products, or a method detecting theactivity of proteins that are gene translation products.

[0098] Compounds isolated by these screenings include, for example, (1)compounds that bind to the protein of the present invention and promoteor inhibit its activity, (2) compounds that bind to the protein of thepresent invention, or ligands of the protein of the present inventionlike Reg protein or the like, and promote or inhibit the binding of theprotein of the present invention and ligands, (3) compounds that bind toligands of the protein of the present invention and promote or inhibittheir activation, and (4) compounds that promote or inhibit the signaltransduction from the protein of the present invention to the expressionof a changes of cells.

[0099] Such compounds can be applied as preventive or therapeutic agentsagainst diseases caused by disorders of signal transduction systems thatmediate the protein of the present invention (for example, diseasescaused by functional disorders of pancreatic β cells). For example,these compounds can be applied as therapeutic agents for diabetes.

[0100] DNAs of the present invention, proteins of the present inventionor partial peptides thereof, vectors comprising DNAs of the presentinvention, antibodies against the protein of the present invention orpartial peptides thereof, and compounds isolated by the screeningsdescribed above, can be used alone, or as a combination with othercompounds when using as therapeutic agents. Reagents and drugs areincluded in the therapeutic agent of the present invention.

[0101] For example, since the protein of the present invention has abinding activity towards Reg protein, the protein of the presentinvention and partial peptides thereof can be used for the binding toReg protein. Such proteins or peptides can be used for the detection ofthe Reg protein or for affinity purification. By contacting the proteinof the present invention or partial peptides thereof with the Regprotein, the protein of the present invention or partial peptidesthereof can be bound to the Reg protein. The protein of the presentinvention or partial peptides thereof may have been purified orexpressed on the cell membrane surface. They can also be bound tocarriers. There is no limitation on the origin of the Reg protein to bebound, and the mouse, rat, or human Reg protein can be used. Moreover,DNAs encoding the protein of the present invention or partial peptidesthereof, and vectors to which the DNAs have been inserted can be usedfor the same purpose by expressing the protein of the present inventionor partial peptides thereof in the cells. Thus, the protein of thepresent invention or partial peptides thereof, DNAs encoding them, ortherapeutic agents comprising vectors carrying the DNAs can be Regprotein-binding agents.

[0102] Moreover, the protein of the present invention functions as a Regprotein receptor. Therefore, the protein of the present invention can beused for the regulation (promotion or suppression) of intracellularsignal transduction in response to the Reg protein. By activating theprotein of the present invention, the signal transduction is promoted,and inversely, by inhibiting the activation, signal transduction isblocked. For example, by contacting cells expressing the protein of thepresent invention (for example, SEQ ID NO: 4) with ligands of theprotein of the present invention such as Reg protein, or agonists, theprotein of the present invention is activated and signals are transducedto the cell interior. Cells are preferably of pancreatic β cell lineage,epithelial cells, etc. Moreover, proteins that bind to Reg protein, butdo not transduce signals to cell interior, can be used for blocking thesignal transduction of the Reg protein. As an example, a proteincomprising the region binding to Reg protein, but not the region thattransduces signals to the downstream can be given. By expressing suchproteins in the cells, or adding them extracellularly, the signaltransduction by Reg protein can be blocked. DNAs encoding the protein ofthe present invention or partial peptides thereof, and vectors to whichthe DNAs have been inserted can be used for the same purpose, byexpressing the protein of the present invention or partial peptidesthereof in the cells. Moreover, antibodies binding to the protein of thepresent invention or partial peptides thereof, or compounds isolated bythe screenings of the present invention can be used for the samepurpose. Therefore, the protein of the present invention or partialpeptides thereof, DNAs encoding the proteins or peptides, vectors towhich the DNAs have been inserted, antibodies of the present invention,and compounds isolated by the screening of the present invention, can beregarded as regulators (promoters, suppressors, etc.) of intracellularsignal transduction in response to Reg protein.

[0103] Examples of the intracellular signal transduction in response toReg protein are, promotion of cellular DNA synthesis and regulation ofcell growth (promotion or suppression). Namely, this shows that theprotein of the present invention can be used for suppressing cellularDNA synthesis, and promotion or suppression of cell growth. Target cellsare preferably cells of pancreatic β cell lineage, epithelial cells,etc. Cell growth (or cell division) can be promoted by contacting cellsexpressing the protein of the present invention with ligands (forexample, Reg protein) or agonists of the protein of the presentinvention to promote DNA synthesis. When expressing the protein of thepresent invention exogenously in cells, vectors expressing the proteinof the present invention (for example, SEQ ID NO: 4) are introduced intothe cells. Moreover, proteins that bind to Reg protein, but do nottransduce signals to the cell interior can be used for inhibiting DNAsynthesis or cell growth. As an example, a protein comprising the regionbinding to Reg protein, but not the region that transduces signals tothe downstream can be given. DNA synthesis or cell growth can besuppressed by expressing such proteins intracellularly or adding themextracellularly. DNAs encoding the protein of the present invention orpartial peptides thereof, or vectors to which the DNAs have beeninserted, can be used for the same purpose, by expressing the protein ofthe present invention or partial peptides thereof in the cells.Moreover, antibodies binding to the protein of the present invention orpartial peptides thereof, and compounds isolated by the screening of thepresent invention can be used for the regulation of DNA synthesis orcell growth. For example, antibodies or compounds functioning as ligandsor agonists of the protein of the present invention can promote growthof cells (such as pancreatic β cells), by administrating these ligandsand agonists to the living body. The administration can be conducted invitro and in vivo. Thus, the protein of the present invention or partialpeptides thereof, DNAs encoding the protein or peptides, vectors towhich the DNAs have been inserted, antibodies of the present invention,and compounds isolated by the screening of the present invention can beregulators (promoters or suppressors) of cellular DNA synthesis or cellgrowth.

[0104] Moreover, as an example of the signal transduction elicited bythe activation of the protein of the present invention, cell apoptosiscan be given. Namely, the protein of the present invention can be usedfor the regulation of cell apoptosis (induction of apoptosis or thesuppression of the induction). DNAs encoding the protein of the presentinvention or partial peptides thereof, and vectors into which the DNAshave been inserted can be used for the same purpose, by expressing theprotein of the present invention or partial peptides thereof in thecells. Moreover, antibodies binding to the protein of the presentinvention or partial peptides thereof, and compounds isolated by thescreening of the present invention can also be used for the regulationof apoptosis. Target cells are preferably of pancreatic β cell lineage,epithelial cells, etc. Apoptosis can be induced by contacting cellsexpressing a high concentration of the protein of the present inventionwith ligands of the protein of the present invention (for example, Regprotein). Reg protein is contacted with cells at a concentration higherthan 100 nM, preferably 500 nM or more, more preferably 1000 nM or more.In the case of expressing the protein of the present inventionexogenously in the cells, vectors expressing the protein of the presentinvention (for example, SEQ ID NO: 4) are introduced into the cells.Moreover, proteins that bind to Reg protein, but do not transducesignals to the cell interior can be used to suppress apoptosis caused byReg protein. Apoptosis can be suppressed by expressing such proteinsintracellularly or by adding them extracellularly. Thus, the protein ofthe present invention and partial peptides thereof, DNAs encoding theprotein or partial peptides thereof, vectors into which the DNAs havebeen inserted, antibodies of the present invention, and compoundsisolated by the screenings of the present invention can be regulators(inducers or suppressors etc.) of apoptosis of cells.

[0105] The protein of the present invention or partial peptides thereof,DNAs encoding the protein or peptides, vectors into which the DNAs havebeen inserted, antibodies of the present invention, and compoundsisolated by the screenings of the present invention can be made into acomposition by combining with distilled water, a salt, BSA, glycerol, astabilizer, preservative, or detergent, according to well knownpharmacological methods. Moreover, the pharmaceutical agent of thepresent invention can be used as a reagent for pancreatic tests asdescribed above. Moreover, it is also useful as a pharmaceuticalcomposition for treating or preventing diabetes, digestive tract tumors,neurodegeneration diseases, pancreatitis, and other tumors.

[0106] When using the pharmaceutical agent of the present invention as adrug, the protein of the present invention or partial peptides thereof,DNAs encoding the protein or peptides, vectors into which the DNAs havebeen inserted, antibodies of the present invention, and compoundsisolated by the screening of the present invention can be directlyadministered to patients, or can be formulated by a well knownpharmaceutical method. For example, a pharmaceutically acceptablecarrier or medium, specifically, distilled water, physiological saline,dextrose, glycerol, ethanol, vegetable oil, an emulsifying agent,suspension, detergent, stabilizer, and such can be suitably combined forformulation and administered. The pharmaceutical composition of thepresent invention can be in the form of a solution, tablet, capsule,troche, buccal tablet, elixir, suspension, or syrup. The content of theactive compound can be suitably determined. The administration can beconducted, for example, intranasally, transbronchially, intramuscularly,or orally by methods well known to one skilled in the art, in additionto intraarterial, intravenous, or hypodermic injections. Administrationcan be conducted systemically or topically. Dosage changes according tothe weight, age of the patient, administration method, symptoms, andsuch, but a suitable dosage can be appropriately selected by one skilledin the art. Administration can be conducted once or several times.Moreover, as long as the compounds are materials encoded by DNA, genetherapy can be conducted by integrating the DNA into gene therapyvectors. Administration can be conducted ex vivo or in vivo. Theadministration method changes according to the weight, age, symptoms,and such, of the patient, but it can be appropriately selected by oneskilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0107]FIG. 1 shows the result of measuring BrdU incorporation after theaddition of human REG protein (REG Iα) to rat insulinoma derived cellline RINm5F cells (Example 3).

[0108]FIG. 2 shows the result of measuring binding of [¹²⁵I] labeled ratReg protein (Reg I) to RINm5F cells when it is added to the cells(Example 4). “Hot” indicates when only the labeled rat Reg protein isadded, and “Hot+100×Cold” indicates when both labeled rat Reg proteinand 100-folds of non-labeled rat Reg protein is added.

[0109]FIG. 3 shows the result of measuring the binding of [¹²⁵I] labeledrat Reg protein (Reg I) to COS-7 cells expressed with isolatedReg-binding protein when [¹²⁵I] labeled rat Reg protein (Reg I) is addedto the cells (Example 6). “pCI-neo” and “pCI-167.1” indicate resultsfrom cells introduced with empty vector and Reg-binding proteinexpression vector, respectively. Moreover, (−) and (+) indicate resultsin which labeled rat Reg protein only, and both labeled rat Reg proteinand 100 times higher amount of non-labeled rat Reg protein are added,respectively.

[0110]FIG. 4 shows alignment of the predicted protein amino acidsequences of rat Reg receptor (rEXTL3) (SEQ ID NO: 4), human EXTL3/EXTR1(hEXTL3) (GenBank accession numbers AF001690 and AB007042) (SEQ ID NO:5), human EXT2 (hEXT2) (GenBank accession number U64511) (SEQ ID NO: 6),human EXT1 (hEXT1) (GenBank accession number S79639) (SEQ ID NO: 7),human EXTL1 (hEXTL1) (GenBank accession number U67191) (SEQ ID NO: 8),and human EXTL2 (hEXTL2) (GenBank accession number AF000416) (SEQ ID NO:9) (Example 7). The transmembrane domain is underlined. The numbers onthe right correspond to amino acid residues. Residues identical to ratReg-binding protein (rEXTL3) are indicated by dots. Hyphens denote theabsence of corresponding residues in rat Reg-binding protein (rEXTL3).

[0111]FIG. 5 shows the cellular distribution of Reg-binding protein.Lane 1, homogenate of COS-7 cells to which the control vector had beenintroduced; lane 2-6, homogenate, membrane fraction, mitochondrialfraction, microsomal fraction and cytosolic fraction of COS-7 cell(Example 8) into which the Reg receptor expression vector had beenintroduced. Ten μg protein was electrophoresed in each lane, and Westernblot analysis was carried out by using an antibody against the HA tagbinding to Reg-binding protein.

[0112]FIG. 6 shows that the rat homologue of human EXTL3/EXTR1 is a cellsurface type Reg-binding protein (Example 9). This figure shows thebinding of [¹²⁵I] Reg protein to Reg receptor-expressing cells with (+;100-fold excess) or without (−) unlabeled rat Reg protein. “pCIneo” isthe control in which an empty vector has been introduced, and“pCI•rEXTL3” is the result of introducing Reg-binding protein expressionvector to cells. Results are presented as the mean±S.E.M. of 4 separateexperiments.

[0113]FIG. 7 shows the functional characterization of Reg receptor. (A)BrdU incorporation by rat Reg protein into CHO cells stably expressingthe Reg receptor (Example 10). Two independent cell lines expressing theReg receptor (RegR-#3 and RegR-#22) were tested. Results are presentedas the mean±S.E.M. of 8 separate experiments. (B) Competition bindingcurves for rat Reg (circle) and human REG (square) with rat Regreceptor. Results are presented as the mean±S.E.M. of 4 separateexperiments.

[0114]FIG. 8 shows proliferation and apoptosis of Reg receptorexpressing β-cells (Example 11). Three independent cell lines expressingthe Reg receptor (#1, #6 and #24) were tested. RIN is the RINm5Fcontrol. Results are presented as the mean±S.E.M. of 4-8 separateexperiments. (A) BrdU incorporation by rat Reg protein into RINm5F cellsstably expressing the Reg receptor. (B) Cleavage of WST-1 by viablecells was increased by Reg protein. (C) Reg protein-induced apoptosis ofRINm5F cells was quantified by the TUNEL method.

[0115]FIG. 9 shows expression of Reg receptor mRNA (Example 12). RNaseprotection assay was carried out for measuring expression of Regreceptor mRNA. 309 nucleotide band corresponds to the protection size byReg receptor mRNA. (A) Expression of Reg receptor mRNA in β-cells.Regenerating Langerhans' islets were isolated from 90% pancreatectomizedrats receiving intraperitoneal administration of 0.5 mg/kg/daynicotinamide for 1-3 months (K. Terazono, et al., J. Biol. Chem. 263,2111(1988); K. Terazono, T. Watanabe, Y. Yoneyama, in Molecular biologyof the islets of Langerhans', H. Okamoto, Ed. (Cambridge UniversityPress, Cambridge, 1990), pp. 301-313; K. Terazono et al., Diabetologia33, 250(1990); Y. Yonemura et al., Diabetes 33, 401(1984)): Lane 1,normal pancreatic islets; lane 2, regenerating islets one month afterthe partial pancreatectomy: lane 3, regenerating Langerhans' islets twomonth after the partial pancreatectomy: lane 4, regenerating isletsthree month after the partial pancreatectomy: lane 5, RINmSF cells; lane6, ARIP cells. Probe alone was applied in lane P. (B) Expression of Regreceptor mRNA in rat tissues: Lane 1, normal pancreatic islets; lane 2,whole pancreas; lane 3, liver; lane 4, kidney; lane 5, heart; lane 6,spleen; lane 7, thymus; lane 8, testis; lane 9, adrenal gland; lane 10,stomach; lane 11, jejunum; lane 12, ileum; lane 13, colon; lane 14,pituitary gland; lane 15, brain.

[0116]FIG. 10 shows cleavage of WST-1 by viable cells increased by Regprotein in CHO cells stably expressing Reg receptor. Two independentcell lines as in FIG. 7A were used. Results are presented as themean±S.E.M. of 8 separate experiments.

BEST MODE FOR CARRYING OUT THE INVENTION

[0117] Herein below, the present invention is explained specificallyusing examples, but it is not to be construed as being limited thereto.

Example 1 Construction of Expression Vector of Human REG Protein (REGIα) and Rat Reg Protein (Reg I)

[0118] Full length of the protein coding region of human REG Ia cDNA(Terazono, K. et al., J. Biol. Chem. 263, 2111-2114 (1998)) was insertedto SnaBI/AvrII site at the downstream of yeast alcohol oxidase promoterof Pichia expression vector pPIC3.5 (Invitrogen) using a linker toconstruct the expression vector. Full length of the protein codingregion of Rat Reg I cDNA (Terazono, K. et al., described above) was alsoinserted similarly to SnaBI/NotI site of pPIC3.5 using a linker. Thesetwo expression vector DNAs were purified by CsCl method, and introducedto competent cells (Pichia GS115 strain) prepared using Pichia Easy CompKit (Invitrogen) Cells into which the expression vector has beenintroduced were selected by the fact that these cells acquire theability to grow in a medium without histidine. Among these cells, aclone in which the amount of human REG protein or rat Reg proteinproduced and secreted into the medium becomes maximum when methanol isadded was selected.

Example 2 Preparation of Human REG Protein (REG Iα) and Rat Reg Protein(Reg I)

[0119]Pichia (Pichia pastoris) producing human REG protein or rat Regprotein described above was precultured at 28˜30° C. for 16˜18 hours inthe BMGY medium (1% yeast extract, 2% polypeptone, 100 mM potassiumphosphate buffer (pH 6.0), 1.34% Yeast Nitrogen Base, 0.00004% biotin,1% glycerol). Then, it was cultured on a large scale until OD₆₀₀ became2˜5 in the BMGY medium. The yeast were collected by centrifugation, andresuspended in BMMY medium (1% yeast extract, 2% polypeptone, 100 mMpotassium phosphate buffer (pH 6.0), 1.34% Yeast Nitrogen Base, 0.00004%biotin, 0.5% methanol), and cultured at 28˜30° C. for 3˜4 days. Duringthe time, methanol was added to a final concentration of 0.5% atintervals of 24 hours. The culture supernatant was collected and aceticacid was added to adjust pH to 3.5. The pH adjusted culture medium wasapplied to STREAMLINE SP (Pharmacia) equilibrated by 50 mM sodiumacetate (pH 3.5), and after washing with 50 mM sodium acetate (pH 3.5),it was eluted with 50 mM sodium acetate (pH 3.5)/0.5 M NaCl. Massspectrometry was used to confirm that proteins produced were human REGprotein or rat Reg protein.

Example 3 Effects of Addition of REG Protein Toward Rat InsulinomaDerived Cultured Cells, the RINm5F Cells

[0120] REG protein was added to rat insulinoma derived cultured cells,the RINm5F cells (Zenilman, M. E. et al., Gastroenterology 110,1208-1214 (1996)) and the incorporation of 5′-bromo-2′-deoxyuridine(BrdU) (cell growth activity) was measured. First, 5×10⁵ cells/ml ofRINm5F cells were seeded onto 96 well plates at 100 μl/well and culturedfor 2 days at 37° C. After that, the culture medium was changed to 100μl/well of the medium described below. As for Human REG Iα, the onedescribed in Example 2 was used.

[0121] Medium+1% FCS

[0122] Medium+1% FCS+human REG Iα (1 nM; 0.016 μg/ml)

[0123] Medium+1% FCS+human REG Iα (10 nM; 0.16 μg/ml)

[0124] Medium+1% FCS+human REG Iα (100 nM; 1.6 μg/ml)

[0125] Medium+1% FCS+human REG Iα (1000 nM; 16 μg/ml)

[0126] The cells were incubated at 37° C. for 24 hours, and then, 10μl/well of BrdU labeling solution (10 mM BrdU stock solution was dilutedwith medium to be 100 μM) was added (final concentration 10 μM). Afterincubation at 37° C. for 12 hours, medium was removed and 200 μl/well ofFixDenat (Roche Diagnostics) was added. After incubation at roomtemperature for 15 min, FixDenat solution was removed, and then 100μl/well of anti-BrdU-POD antibody ({fraction (1/100)} diluted solutionof stock solution, Roche Diagnostics) was added. After incubating atroom temperature for 60 min, the anti-BrdU-POD antibody solution wasremoved, and then rinsed three times with 200 μl/well of washingsolution (10× washing solution, Roche Diagnostics). 100 μl/well ofsubstrate solution (Roche Diagnostics) was added and incubated at roomtemperature until a sufficient color development was obtained.Absorbance of each sample at 370 nm was measured using an ELISA reader(reference wavelength: approx. 492 nm).

[0127] As a result, REG protein concentration dependent cell growth wasobserved (FIG. 1).

Example 4 Assay of the Binding Activity of Reg Protein Towards RINm5FCells

[0128] First, diluted [¹²⁵I] Reg I solution was prepared as describedbelow. [¹²⁵I] rat Reg I stock solution (50 ng/μl=˜3.33 μm, 8.6×10⁵cpm/μl) was diluted with DMEM to be 1 nM, 333 pM, 100 pM, 33 pM, and 10pM. Moreover, diluted solution with a 100-fold concentration ofnon-labeled Reg I stock solution (460 ng/μl=30.6 μM) was similarlyprepared. 3 ml of 4×10⁵ cells/ml RINm5F cells were seeded onto 6 wellplates and cultured at 37° C. for 2 days. After washing with ice coldDMEM, 3 ml of DMEM containing [¹²⁵I] rat Reg I described above was added(final concentration: 10 pM, 33 pM, 100 pM, 333 pM, and 1 nM). In acompetitive inhibition experiment, DMEM with a 100-fold amount ofnon-labeled Reg I was used. After keeping on ice for 2 hours, the cellswere washed with DMEM for 3 times, and then lysed by adding 0.5˜1ml/well of [100 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1% Triton X-100] and[¹²⁵I] radioactivity was counted by γ-counter.

[0129] As a result, it was seen that the excessive amount of non-labeledReg protein inhibited the binding, indicating the existence of amolecule necessary for specific binding on the cell membrane of RINm5Fcells (FIG. 2).

Example 5 Identification and Isolation of Reg-Binding Protein

[0130] Rat pancreatic Langerhans' islet expression cDNA library wasconstructed by the λZAP II vector using poly (A)+RNA of rat pancreaticLangerhans' islets as template. Rat Reg protein prepared in Example 2was labeled with [¹²⁵I] using Bolton-Hunter reagent, and phage clonesbinding to Reg protein was selected and isolated by West-Western methodfrom the expressed cDNA library.

[0131] Recombination of cDNA into a plasmid vector (pBluescript SK(−)Stratagene) was carried out by an in vivo excision method using helperphage from positive phage clones. Nucleotide sequence of cDNA wasdetermined by the dideoxy method. The nucleotide sequence and expectedamino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2,respectively. The Protein estimated from the nucleotide sequence wasthought to be a cell membrane protein with a transmembrane domaincomprising a hydrophobic amino acid cluster.

Example 6 Expression of Reg-Binding Protein in COS-7 Cells

[0132] cDNA isolated in Example 5 was integrated into a mammalian cellexpression vector comprising a cytomegalovirus promoter (pCI-neo)(Promega) to construct a Reg-binding protein expression vector(pCI-167.1). The vector was introduced into COS-7 cells byelectroporation method and expressed transiently. 48 hours afterintroducing the vector, Reg binding activity was examined by a protocolsimilar to that described in Example 4.

[0133] Specifically, first, [¹²⁵I] rat Reg I stock solution (50ng/μl=˜3.33 μM, 2.7×10⁵ cpm/μl) was diluted to 10 nM using DMEM. Inaddition, a diluted solution in which non-labeled Reg I stock solution(2250 ng/μl=150 μM) was added at 100-folds the concentration of [¹²⁵I]Reg I (1 μm), was prepared.

[0134] 3 ml of 2.5×10⁵ cells/ml transfected COS cells were seeded onto 6well plates and cultured at 37° C. for 2 days. After washing withice-cold DMEM, 3 ml DMEM containing [¹²⁵I] rat Reg I was added (10 nM,final concentration). In a competitive inhibition experiment, 100-foldamount of non-labeled Reg I coexisted. After keeping on ice for 2 hoursand then washing 3 times with DMEM, 1 ml/well of [100 mM Tris-HCl (pH7.6), 1 mM EDTA, 1% Triton X-100] was added to lyze the cells, and[¹²⁵I] radioactivity was counted by using a γ-counter.

[0135] As a result, the binding to [¹²⁵I] labeled Reg protein increasedsignificantly in cDNA-introduced cells, in comparison with the cells towhich only the vector was introduced. Moreover, the binding disappearedby the addition of an excessive amount of non-labeled Reg protein (FIG.3). Therefore, it was thought that the protein encoded by the isolatedcDNA was a molecule binding to Reg protein on a mammalian cell membrane,and that it can be a receptor molecule which plays a key role in β cellregeneration and proliferation activity of Reg protein.

Example 7 Screening of Rat Pancreatic Islet cDNA Library

[0136] To further isolate cDNA encoding a Reg-binding protein, a ratislet cDNA library (5×10⁶ clones) was screened by plaque hybridizationusing the cDNA fragment obtained in Example 5 as probe, and 8 positiveclones were obtained. The 8 clones largely overlapped with each otherand had complete nucleotide identity in the overlapping regions. Theobtained cDNA sequence encoding rat Reg-binding protein and amino acidsequence of Reg-binding protein encoded by the cDNA are shown in SEQ IDNO: 3 and SEQ ID NO: 4, respectively.

[0137] As shown in FIG. 4, the cDNA has a 2,760 bp open reading frameencoding a 919 amino acid protein, and the deduced amino acid sequenceof the cDNA predicted that the protein is a type II transmembrane domainwith a long extracellular domain (868 amino acid residues), atransmembrane domain (residues 29-51) and a short intracellular regionat the N-terminus.

Example 8 Expression of Rat Reg-Binding Protein

[0138] An expression vector for the rat Reg protein cDNA isolated inExample 7 was constructed, and it was transiently expressed in COS-7cells. The rat Reg binding protein cDNA, into which an oligonucleotideencoding hemagglutinin (HA) nonapeptide-tag (YPYDVPDYA) at theN-terminus was ligated, was inserted into a pCl-neo mammalian expressionvector (Promega). This vector was introduced to COS-7 cells byelectroporation and expressed. After a 48 h incubation, cells werecollected, homogenized, and fractionated as described (S. Takasawa etal., J. Biol. Chem. 268, 26052 (1983); H. Okamoto et al., Meth. Enzymol.280, 306 (1997)). The protein sample was electrophoresed on a 12.5%(w/v) SDS-polyacrylamide gel and transferred to immobilon-P (Millipore).Western blot analysis was carried out described as in S. Takasawa etal., J. Biol. Chem. 270, 30257 (1995); H. Okamoto et al., Meth. Enzymol.280, 306 (1997). Monoclonal antibody against HA was anti-HA 3F10(Boehringer).

[0139] Immunoblot analysis revealed that the protein encoded by the cDNAwas expressed predominantly in the cell membrane fraction with anapparent molecular weight of 105 kD (FIG. 5A), coinciding with themolecular weight calculated from the presumed amino acid sequence.(104,682).

Example 9 Binding of Rat Reg-Binding Protein to Reg Protein

[0140] The rat Reg-binding protein (also called EXTL3/EXTR1) expressionvector constructed in Example 8 or the control vector was introducedinto COS-7 cells by electroporation and expressed transiently. CHO cellsexpressing the Reg receptor stably were isolated as described above. Thecells (7.5×10⁵ cells) were washed with RPMI1640 (Roswell Park Memorialinstitute 1640 medium) and incubated on ice in the presence of ¹²⁵Ilabeled rat Reg protein (50 ng/ml, 1.5×10⁵ cpm/ml) with variousconcentrations of unlabeled rat Reg or human REG protein in RPMI1640containing 1% fetal calf serum for 2 h. After washing with RPMI1640three times, cells were solubilized by 1 ml of 100 mM Tris-HCl (pH 7.6),1 mM EDTA and 1% Triton X-100. The radioactivity of the lysatewas-determined by a γ-counter (Cobra, Packard). As a result, rat Regbinding protein expression vector-introduced-COS-7 cells bound to¹²⁵I-labeled rat Reg protein and the binding was decreased by theaddition of unlabeled Reg protein (FIG. 6).

[0141] A homology search against DNA and protein databases revealed thatthe cDNA of rat Reg-binding protein (SEQ ID NO: 3) and its deduced aminoacid sequence (SEQ ID NO: 4) shows significant homologies to those ofmultiple exostoses (EXT) family genes, especially to human EXT-like gene3 (EXTL3)/EXT-related gene 1 (EXTR1) (W. Van Hui et al., Genomics 47,230(1998); T. Saito et al., Biochem. Biophys. Res. Commun. 243, 61(1998)) (over 97% amino acid identity), indicating that the cDNA encodesa rat homologue to human EXTL3/EXTR1. The EXTL3/EXTR1 gene has beenisolated as a member of the EXT family genes by homology screening, butits physiological function and pathological significance have not yetbeen clarified. EXTL3/EXTR1 is thought to belong to the EXT family (W.Van Hui et al., Genomics 47, 230 (1998); T. Saito et al., Biochem.Biophys. Res. Commun. 243, 61 (1998)) because it shows homology to EXT2and EXT1 at their C-terminal regions (52% in C-terminal 262 amino acidswith EXT2 and 40% in C-terminal at 247 amino acids with EXT1) (see FIG.1). However, the N-terminal region (residues 1-656) of EXTL3/EXTR1 hasno homology to any other members of the EXT family genes. Furthermore,the N-terminal region of EXTL3/EXTR1 contained a transmembrane domain,but the other members of the family lacked this domain, and therefore,were not thought to be cell surface proteins. In addition, the 1.6 kbpcDNA, which was initially isolated in the screening of the rat isletcDNA expression library as a Reg-binding protein, contained only theN-terminal region (amino acid residues 1-332). Therefore, it isreasonable to assume that the N-terminal region contains the Reg bindingdomain and that the EXT family members other than EXTL3/EXTR1 have noability to bind to Reg protein.

Example 10 Stimulation Effect of Rat Reg-Binding Protein (Rat RegReceptor) Expression Cell by Reg Protein

[0142] The expression vector constructed in Example 8 was introducedinto CHO cells and several cell lines overexpressing the receptorprotein were established, and 5′-bromo-2′-deoxyuridine (BrdU)incorporation into the cells in response to rat Reg protein stimulationwas examined.

[0143] The rat receptor expression vector with HA-tag was introducedinto CHO cells and RINm5F cells. Cells were cultured in Roswell ParkMemorial Institute 1640 medium (RPMI1640) with 10% fetal calf serum (BioWhittaker, Walkersville, Md.) and 250 μg/ml neomycin (Gibco) for 2 weeks[S. Takasawa et al., J. Biol. Chem. 273, 2497 (1998)]. Stabletransformants expressing high levels of the recombinant protein werescreened by immunoblot analysis of HA and isolated. Stable transformantsexpressing Reg receptor were cultured in RPMI1640 medium with 1% fetalcalf serum in the presence of increasing concentrations of rat Regprotein for 24 h. During the last 2 h, BrdU (10 μM) was added to theculture medium and BrdU incorporation was measured using a colorimetriccell proliferation ELISA kit (Boehringer).

[0144] The BrdU incorporation of EXTL3/EXTR1 expressing cell lines (both#3 and #22) was significantly increased when incubated with 1-300 nM ratReg protein (EC₅₀=4.01 nM in line #3 and 1.11 nM in line #22, FIG. 7A).The Reg protein concentrations exhibiting growth-stimulating effects onthe CHO-cell lines were consistent with those for primary cultured ratislets (T. Watanabe et al., Proc. Natl. Acad. Sci. USA 91, 3589 (1994)),suggesting that the replication of pancreatic β-cells by Reg protein ismediated by the rat homologue to EXTL3/EXTR1.

[0145]¹²⁵I-labeled rat Reg protein bound to the CHO cells (K_(d)=4.41nM) and the binding was displaced by increasing the concentrations ofunlabeled rat Reg protein (K_(i)=1.61 nM; FIG. 7B) (refer to Example 9).The Hill coefficient for rat Reg protein was estimated to be n_(H)=1.18,indicative of interactions with a single, homogenous population ofbinding sites. In addition, human REG protein (K. Terazono, et al., J.Biol. Chem. 263, 2111 (1988); K. Terazono, T. Watanabe, Y. Yonemura, inMolecular biology of the islets of Langerhans', H. Okamoto, Ed.(Cambridge University Press, Cambridge, 1990), pp. 301-313), which showsa 70% amino acid identity to rat Reg protein, also displaced the bindingof radio-labeled rat Reg protein and CHO cells, but the displacementrequired higher concentrations (K_(i)=7.41 nM; FIG. 7B). These resultsstrongly suggest that EXTL3/EXTR1 is a cell surface Reg receptor thatbinds to Reg protein and transduces the growth stimulating signals ofReg protein.

Example 11 Functional Analysis of Rat Reg Receptor

[0146] Reg is recognized as a β-cell growth factor (H. Okamoto, J. Mol.Med. 77, 74 (1999); T. Watanabe et al., Proc. Natl acad. Sci. USA 91,3589 (1994); D. J. Gross et al., Endocrinology 139, 2369 (1998)). WhenReg protein was added to rat insulinoma derived β cell line RINm5F, BrdUincorporation of the cells increased (1.5˜2 fold) showing that itstimulates increase of cell number Reg proteinconcentration-dependently. It was suggested that since Reg proteinconcentration stimulating the growth of RINm5F cells coincides with thatin rat pancreatic islet primary culture, Reg protein may react throughthe same receptor in both cells. Next, expression vector constructed inExample 8 was introduced to RINm5F cells to establish several Regreceptor overexpressing cell lines, and using these cell lines thefunction of Reg protein was examined.

[0147] The BrdU incorporation (refer to Example 10 for assay method) ofthe receptor expressing cell lines (lines #1, #6 and #24) wassignificantly increased when incubated with 0.3˜300 nM rat Reg protein(FIG. 8A).

[0148] After a 24 h incubation of the stable transformants expressingReg receptor in RPMI1640 medium with 1% fetal calf serum in the presenceof various concentrations of rat Reg protein, a solution containingWST-1 was added to the medium and cultured further for 30 min and thecleavage of tetrazolium salt4[-3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate (WST-1) by mitochondrial dehydrogenases was measuredinviable cells using a Cell Proliferation Reagent WST-1 (Boehringer).The cell number of RINm5F cells were increased in response to theaddition of Reg protein (0.3-100 nM), but were reduced when the cellswere incubated with high concentrations of Reg protein (FIG. 8B).

[0149] To evaluate the possibility that a high-concentration of Regprotein induces apoptosis of these cells, this stable transformantexpressing Reg receptor was incubated for 24 hr in RPMI1640 medium with1% fetal calf serum in the presence of increasing concentrations of ratReg protein. After incubation, apoptosis was detected by the TUNELmethod (Y. Gavrieli, Y. Sherman, S. A. Ben-Sasson, J. Cell Biol. 119,493 (1992)) using an Apoptosis Screening Kit (Wako, Osaka, Japan) By theapoptosis assay of these cells, it was revealed that the highconcentration of Reg protein induced apoptosis of Reg receptorexpressing RINm5F cells (FIG. 8C). These results indicate that the Regreceptor mediates the proliferation and apoptosis of pancreatic β-cellsin response to Reg protein, thereby maintaining a stable β-cell mass.

Example 12 Expression Assay of Reg Receptor mRNA

[0150] The expression of the Reg receptor mRNA was examined in variousrat tissues and cells by RNase protection assay.

[0151] Rat regenerating pancreatic islets were prepared as describedbefore (K. Terazono, et al., J. Biol. Chem. 263, 2111 (1988); K.Terazono, T. Watanabe, Y. Yonemura, in Molecular biology of the isletsof Langerhans', H. Okamoto, Ed. (Cambridge University Press, Cambridge,1990), pp. 301-313; Y. Yonemura et al., Diabetes 33, 401 (1984)). RNAswere isolated from various rat tissues and cell lines as describedbefore (T. Koguma et al., Biochem. Biophys. Acta 1223, 180 (1994); N.Noguchi et al., J. Biol. Chem.272, 3133 (1997) H. Okamoto et al. Meth.Enzymol. 280, 306 (1997)). The Pst I/Bgt II fragment of rat Reg receptorcDNA was subcloned into the Pst I/Bam HI site of pBluescript SK (−),linearized with Hind III and transcribed in vitro by T3 RNA polymeraseusing [α-³²P] CTP. The resultant 0.45 kb cRNA was used as a probe. RNaseprotection assay was performed using an RPA III kit (Ambion) accordingto instructions.

[0152] As shown in FIG. 9A, the Reg receptor mRNA was expressed innormal pancreatic islets, regenerating pancreatic islets and RINm5Fβ-cells. The expression of the Reg receptor was not increased inregenerating pancreatic islets as compared to that in normal pancreaticislets, suggesting that the regeneration and proliferation of pancreaticβ-cells that increases β-cell mass is primarily regulated by theexpression of Reg protein but not by the expression of the receptor.This hypothesis is consistent with the observations that Reg gene wasfirst identified as a gene specifically expressed in regeneratingpancreatic islets (K. Terazono, et al., J. Biol. Chem. 263, 2111 (1988);K. Terazono, T. Watanabe, Y. Yonemura, in Molecular biology of theislets of Langerhans', H. Okamoto, Ed. (Cambridge University Press,Cambridge, 1990), pp. 301-313; K. Terazono et al., Diabetologia 33, 250(1990)) and that Reg gene expression was also observed in the phase oftransient β-cell proliferation such as in pancreatic islets ofBB/Wor/Tky rats during the remission phase of diabetes (C. Ishii et al.,Endocr. J. 40, 269 (1993)), pancreatic islets of NOD mice during activediabetogenesis (N. J. Baeza et al., Diabetes 45, 67 (1996)) andpancreatic ductal cells (which are thought to be progenitor cells ofβ-cells), during differentiation and proliferation in a mouse model ofautoimmune diabetes (E. Anastasi et al., Eur. J. Endocrinol. 141, 644-52(1999)). ARIP cells, a pancreatic ductal cell line, which express theReg receptor (see FIG. 9A, lane 6), were also reported to proliferate ina Reg protein-dependent manner (M. E. Zenilman et.al., Gastroenterology110, 1208 (1996); M. E. Zenilman et al., Pancreas 17, 256 (1998)). Theexpression of Reg receptor mRNA was also detected in liver, kidney,stomach, small intestine, colon, adrenal gland, pituitary gland andbrain, but not in heart (FIG. 9B), suggesting the possible involvementof the Reg-Reg receptor signal system as a control mechanism of cellproliferation and apoptosis in a variety of cell-types other thanpancreatic β-cells. In fact, Reg receptor expressing CHO cell linesproliferated in response to Reg protein (FIG. 7A). Furthermore, the CHOcells increased and decreased in number (refer to Example 11 for assaymethod) depending on the Reg protein concentration (FIG. 10).

INDUSTRIAL APPLICABILITY

[0153] The present invention provides a Reg-binding protein (Regreceptor). Reg protein is a cell growth factor for pancreatic β cells,and it is known that it exerts cell growth activity in epithelial cells,and such, as well. It is thought that the Reg-binding protein has thefunction of transducing signals required for cell growth by binding withReg protein in pancreatic β cells, and that pancreatic β cellsregenerate through the binding of Reg protein and Reg-binding protein.Therefore, by analyzing the structure of the extracellular domain ofReg-binding protein and searching analogs of the ligand binding to thedomain, it is possible to produce “anti-diabetic therapeutic agents”inducing physiological growth of pancreatic β cells. Moreover, since Regprotein does not cause overgrowth of β cells in the pancreas, it isthought that the possibility of causing hypoglycemia, as do overdoses ofinsulin, does not exist.

1 10 1 1599 DNA Rattus norvegicus CDS (168)..(1259) 1 tcagcgaggaaaatgaaatt cccattttat ttggtgcctt gtgcagggag cacactgatc 60 cctctagaaccttgtgtgtg aaaaagaggt cgagttttgt caaacagact catggttatg 120 gcaagtgatccgacgtgacc agagtgggca agagccacag tgaactc atg aca ggc 176 Met Thr Gly 1tat acc atg ttg cgg aat ggg gga gtg ggg aac ggt ggt cag acc tgt 224 TyrThr Met Leu Arg Asn Gly Gly Val Gly Asn Gly Gly Gln Thr Cys 5 10 15 atgctg cgc tgg tcc aac cgc atc cgg ctg acc tgg ctg agt ttc acg 272 Met LeuArg Trp Ser Asn Arg Ile Arg Leu Thr Trp Leu Ser Phe Thr 20 25 30 35 ctgttc atc atc ctg gtc ttc ttc ccc ctc att gcc cac tat tac ctc 320 Leu PheIle Ile Leu Val Phe Phe Pro Leu Ile Ala His Tyr Tyr Leu 40 45 50 acc actctg gat gag gca gat gag gcc ggc aag cgc atc ttt ggc ccc 368 Thr Thr LeuAsp Glu Ala Asp Glu Ala Gly Lys Arg Ile Phe Gly Pro 55 60 65 cgg gct ggcaac gag ctc tgt gag gta aag cac gtc cta gat ctt tgt 416 Arg Ala Gly AsnGlu Leu Cys Glu Val Lys His Val Leu Asp Leu Cys 70 75 80 cgg atc cgc gagtct gtg agc gaa gag ctt cta cag cta gaa gcc aag 464 Arg Ile Arg Glu SerVal Ser Glu Glu Leu Leu Gln Leu Glu Ala Lys 85 90 95 cgg cag gag ctg aacagc gag att gcc aag cta aac ctc aag att gaa 512 Arg Gln Glu Leu Asn SerGlu Ile Ala Lys Leu Asn Leu Lys Ile Glu 100 105 110 115 gcc tgt aag aagagt ata gag aac gcc aag cag gac ctg ctg cag ctc 560 Ala Cys Lys Lys SerIle Glu Asn Ala Lys Gln Asp Leu Leu Gln Leu 120 125 130 aag aat gtc attagc cag aca gag cac tcc tac aag gag ctg atg gcc 608 Lys Asn Val Ile SerGln Thr Glu His Ser Tyr Lys Glu Leu Met Ala 135 140 145 cag aac cag cccaaa ctg tca ctg ccc atc cgg ctg ctc cct gag aag 656 Gln Asn Gln Pro LysLeu Ser Leu Pro Ile Arg Leu Leu Pro Glu Lys 150 155 160 gat gac gct ggcctt cca ccc ccc aag gtc act cgg ggt tgc cgg cta 704 Asp Asp Ala Gly LeuPro Pro Pro Lys Val Thr Arg Gly Cys Arg Leu 165 170 175 cac aac tgc ttcgat tac tct cgt tgc cct ctg acg tct ggc ttt cct 752 His Asn Cys Phe AspTyr Ser Arg Cys Pro Leu Thr Ser Gly Phe Pro 180 185 190 195 gtc ttc gtctat gac agt gac cag ttt gcc ttt ggg agc tac ctg gac 800 Val Phe Val TyrAsp Ser Asp Gln Phe Ala Phe Gly Ser Tyr Leu Asp 200 205 210 cct ttg gtcaag cag gct ttt cag gct aca gtg aga gcc aac gtt tat 848 Pro Leu Val LysGln Ala Phe Gln Ala Thr Val Arg Ala Asn Val Tyr 215 220 225 gtt aca gaaaat gca gcc atc gcc tgc ctg tat gtg gtg tta gtg gga 896 Val Thr Glu AsnAla Ala Ile Ala Cys Leu Tyr Val Val Leu Val Gly 230 235 240 gag ata caagag ccc gct gtg ctg cag cct gcc gac ctt gag aag cag 944 Glu Ile Gln GluPro Ala Val Leu Gln Pro Ala Asp Leu Glu Lys Gln 245 250 255 ctg cat tctctg cca cac tgg agg aca gac gga cac aac cat gtc atc 992 Leu His Ser LeuPro His Trp Arg Thr Asp Gly His Asn His Val Ile 260 265 270 275 atc aatctg tcc cgg aag tca gac aca caa aat tta ctg tac aat gtc 1040 Ile Asn LeuSer Arg Lys Ser Asp Thr Gln Asn Leu Leu Tyr Asn Val 280 285 290 agt acaggt cgg gcc atg gtg gcc cag tct acc ttc tat gct gcc cag 1088 Ser Thr GlyArg Ala Met Val Ala Gln Ser Thr Phe Tyr Ala Ala Gln 295 300 305 tac agagct ggc ttt gac ttg gtt gtg tca cca ctt gtc cat gcc atg 1136 Tyr Arg AlaGly Phe Asp Leu Val Val Ser Pro Leu Val His Ala Met 310 315 320 tct gaaccc aac ttc atg gaa atc cca cgt gta act att ttt tca ctt 1184 Ser Glu ProAsn Phe Met Glu Ile Pro Arg Val Thr Ile Phe Ser Leu 325 330 335 ggg agaggt gag gaa gaa caa gag aag ctg ggg gtg tgg aga ggc aga 1232 Gly Arg GlyGlu Glu Glu Gln Glu Lys Leu Gly Val Trp Arg Gly Arg 340 345 350 355 cccccc cca ggc tgg ggt gct ggc ccc tagactaggg tgctgacccc 1279 Pro Pro ProGly Trp Gly Ala Gly Pro 360 tgggctgggg tgctgcgtgc tacctcccac tgtgaaatcgatggtgctca caattgtctc 1339 ttgtaatgta tgtgattttt ttttaaggag aaaaagaaactatttaagat tctgaaggtg 1399 ctactatttt tgttgccaca ggctttaaag aaactttctgagtgggtggg gccttgccca 1459 cttatctttc tctcctccaa atgaggagtt aaaaatgttactaaattgcc cgcacgtgta 1519 atccgctgaa aagaaaaaaa aaaaagaaaa aaaaaaggaaggaaagaagg aaagaaggaa 1579 ggaaggaagg aaggaaagga 1599 2 364 PRT Rattusnorvegicus 2 Met Thr Gly Tyr Thr Met Leu Arg Asn Gly Gly Val Gly Asn GlyGly 1 5 10 15 Gln Thr Cys Met Leu Arg Trp Ser Asn Arg Ile Arg Leu ThrTrp Leu 20 25 30 Ser Phe Thr Leu Phe Ile Ile Leu Val Phe Phe Pro Leu IleAla His 35 40 45 Tyr Tyr Leu Thr Thr Leu Asp Glu Ala Asp Glu Ala Gly LysArg Ile 50 55 60 Phe Gly Pro Arg Ala Gly Asn Glu Leu Cys Glu Val Lys HisVal Leu 65 70 75 80 Asp Leu Cys Arg Ile Arg Glu Ser Val Ser Glu Glu LeuLeu Gln Leu 85 90 95 Glu Ala Lys Arg Gln Glu Leu Asn Ser Glu Ile Ala LysLeu Asn Leu 100 105 110 Lys Ile Glu Ala Cys Lys Lys Ser Ile Glu Asn AlaLys Gln Asp Leu 115 120 125 Leu Gln Leu Lys Asn Val Ile Ser Gln Thr GluHis Ser Tyr Lys Glu 130 135 140 Leu Met Ala Gln Asn Gln Pro Lys Leu SerLeu Pro Ile Arg Leu Leu 145 150 155 160 Pro Glu Lys Asp Asp Ala Gly LeuPro Pro Pro Lys Val Thr Arg Gly 165 170 175 Cys Arg Leu His Asn Cys PheAsp Tyr Ser Arg Cys Pro Leu Thr Ser 180 185 190 Gly Phe Pro Val Phe ValTyr Asp Ser Asp Gln Phe Ala Phe Gly Ser 195 200 205 Tyr Leu Asp Pro LeuVal Lys Gln Ala Phe Gln Ala Thr Val Arg Ala 210 215 220 Asn Val Tyr ValThr Glu Asn Ala Ala Ile Ala Cys Leu Tyr Val Val 225 230 235 240 Leu ValGly Glu Ile Gln Glu Pro Ala Val Leu Gln Pro Ala Asp Leu 245 250 255 GluLys Gln Leu His Ser Leu Pro His Trp Arg Thr Asp Gly His Asn 260 265 270His Val Ile Ile Asn Leu Ser Arg Lys Ser Asp Thr Gln Asn Leu Leu 275 280285 Tyr Asn Val Ser Thr Gly Arg Ala Met Val Ala Gln Ser Thr Phe Tyr 290295 300 Ala Ala Gln Tyr Arg Ala Gly Phe Asp Leu Val Val Ser Pro Leu Val305 310 315 320 His Ala Met Ser Glu Pro Asn Phe Met Glu Ile Pro Arg ValThr Ile 325 330 335 Phe Ser Leu Gly Arg Gly Glu Glu Glu Gln Glu Lys LeuGly Val Trp 340 345 350 Arg Gly Arg Pro Pro Pro Gly Trp Gly Ala Gly Pro355 360 3 3198 DNA Rattus norvegicus CDS (5)..(2761) 3 actc atg aca ggctat acc atg ttg cgg aat ggg gga gtg ggg aac ggt 49 Met Thr Gly Tyr ThrMet Leu Arg Asn Gly Gly Val Gly Asn Gly 1 5 10 15 ggt cag acc tgt atgctg cgc tgg tcc aac cgc atc cgg ctg acc tgg 97 Gly Gln Thr Cys Met LeuArg Trp Ser Asn Arg Ile Arg Leu Thr Trp 20 25 30 ctg agt ttc acg ctg ttcatc atc ctg gtc ttc ttc ccc ctc att gcc 145 Leu Ser Phe Thr Leu Phe IleIle Leu Val Phe Phe Pro Leu Ile Ala 35 40 45 cac tat tac ctc acc act ctggat gag gca gat gag gcc ggc aag cgc 193 His Tyr Tyr Leu Thr Thr Leu AspGlu Ala Asp Glu Ala Gly Lys Arg 50 55 60 atc ttt ggc ccc cgg gct ggc aacgag ctc tgt gag gta aag cac gtc 241 Ile Phe Gly Pro Arg Ala Gly Asn GluLeu Cys Glu Val Lys His Val 65 70 75 cta gat ctt tgt cgg atc cgc gag tctgtg agc gaa gag ctt cta cag 289 Leu Asp Leu Cys Arg Ile Arg Glu Ser ValSer Glu Glu Leu Leu Gln 80 85 90 95 cta gaa gcc aag cgg cag gag ctg aacagc gag att gcc aag cta aac 337 Leu Glu Ala Lys Arg Gln Glu Leu Asn SerGlu Ile Ala Lys Leu Asn 100 105 110 ctc aag att gaa gcc tgt aag aag agtata gag aac gcc aag cag gac 385 Leu Lys Ile Glu Ala Cys Lys Lys Ser IleGlu Asn Ala Lys Gln Asp 115 120 125 ctg ctg cag ctc aag aat gtc att agccag aca gag cac tcc tac aag 433 Leu Leu Gln Leu Lys Asn Val Ile Ser GlnThr Glu His Ser Tyr Lys 130 135 140 gag ctg atg gcc cag aac cag ccc aaactg tca ctg ccc atc cgg ctg 481 Glu Leu Met Ala Gln Asn Gln Pro Lys LeuSer Leu Pro Ile Arg Leu 145 150 155 ctc cct gag aag gat gac gct ggc cttcca ccc ccc aag gtc act cgg 529 Leu Pro Glu Lys Asp Asp Ala Gly Leu ProPro Pro Lys Val Thr Arg 160 165 170 175 ggt tgc cgg cta cac aac tgc ttcgat tac tct cgt tgc cct ctg acg 577 Gly Cys Arg Leu His Asn Cys Phe AspTyr Ser Arg Cys Pro Leu Thr 180 185 190 tct ggc ttt cct gtc ttc gtc tatgac agt gac cag ttt gcc ttt ggg 625 Ser Gly Phe Pro Val Phe Val Tyr AspSer Asp Gln Phe Ala Phe Gly 195 200 205 agc tac ctg gac cct ttg gtc aagcag gct ttt cag gct aca gtg aga 673 Ser Tyr Leu Asp Pro Leu Val Lys GlnAla Phe Gln Ala Thr Val Arg 210 215 220 gcc aac gtt tat gtt aca gaa aatgca gcc atc gcc tgc ctg tat gtg 721 Ala Asn Val Tyr Val Thr Glu Asn AlaAla Ile Ala Cys Leu Tyr Val 225 230 235 gtg tta gtg gga gag ata caa gagccc gct gtg ctg cag cct gcc gac 769 Val Leu Val Gly Glu Ile Gln Glu ProAla Val Leu Gln Pro Ala Asp 240 245 250 255 ctt gag aag cag ctg cat tctctg cca cac tgg agg aca gac gga cac 817 Leu Glu Lys Gln Leu His Ser LeuPro His Trp Arg Thr Asp Gly His 260 265 270 aac cat gtc atc atc aat ctgtcc cgg aag tca gac aca caa aat tta 865 Asn His Val Ile Ile Asn Leu SerArg Lys Ser Asp Thr Gln Asn Leu 275 280 285 ctg tac aat gtc agt aca ggtcgg gcc atg gtg gcc cag tct acc ttc 913 Leu Tyr Asn Val Ser Thr Gly ArgAla Met Val Ala Gln Ser Thr Phe 290 295 300 tat gct gcc cag tac aga gctggc ttt gac ttg gtt gtg tca cca ctt 961 Tyr Ala Ala Gln Tyr Arg Ala GlyPhe Asp Leu Val Val Ser Pro Leu 305 310 315 gtc cat gcc atg tct gaa cccaac ttc atg gaa atc cca ccg cag gtg 1009 Val His Ala Met Ser Glu Pro AsnPhe Met Glu Ile Pro Pro Gln Val 320 325 330 335 cca gtt aag cgg aaa tatctc ttc act ttc cag ggt gag aag att gag 1057 Pro Val Lys Arg Lys Tyr LeuPhe Thr Phe Gln Gly Glu Lys Ile Glu 340 345 350 tct cta aga tct agc cttcag gag gcc cgt tcc ttt gag gaa gaa atg 1105 Ser Leu Arg Ser Ser Leu GlnGlu Ala Arg Ser Phe Glu Glu Glu Met 355 360 365 gag ggt gac cct ccg gccgac tat gat gat cga atc att gcc acc ctc 1153 Glu Gly Asp Pro Pro Ala AspTyr Asp Asp Arg Ile Ile Ala Thr Leu 370 375 380 aag gcc gta cag gac agcaag cta gat cag gtg ctg gta gaa ttt act 1201 Lys Ala Val Gln Asp Ser LysLeu Asp Gln Val Leu Val Glu Phe Thr 385 390 395 tgc aaa aac cag cca aagccc agt ctg cct act gag tgg gca ctg tgt 1249 Cys Lys Asn Gln Pro Lys ProSer Leu Pro Thr Glu Trp Ala Leu Cys 400 405 410 415 ggg gag cgg gag gaccgg cta gag tta ctg aag ctc tcc acc ttc gcc 1297 Gly Glu Arg Glu Asp ArgLeu Glu Leu Leu Lys Leu Ser Thr Phe Ala 420 425 430 ctc atc atc act cccggg gac ccg agc ctg ctt atc tca tct ggc tgt 1345 Leu Ile Ile Thr Pro GlyAsp Pro Ser Leu Leu Ile Ser Ser Gly Cys 435 440 445 gca aca cgg ctc tttgaa gcc ttg gag gtg gga gct gtg cct gtt gtc 1393 Ala Thr Arg Leu Phe GluAla Leu Glu Val Gly Ala Val Pro Val Val 450 455 460 ctt ggg gag cag gtgcag ctt ccg tac cac gac atg cta caa tgg aat 1441 Leu Gly Glu Gln Val GlnLeu Pro Tyr His Asp Met Leu Gln Trp Asn 465 470 475 gag gcc gcc cta gtggtg ccc aag cct cgt gtt aca gag gtt cac ttc 1489 Glu Ala Ala Leu Val ValPro Lys Pro Arg Val Thr Glu Val His Phe 480 485 490 495 ctg tta cga agtctg tca gac agt gat ctg ttg gct atg agg cgg caa 1537 Leu Leu Arg Ser LeuSer Asp Ser Asp Leu Leu Ala Met Arg Arg Gln 500 505 510 ggc cgc ttt ctctgg gag acc tac ttc tcc acc gct gac agt att ttt 1585 Gly Arg Phe Leu TrpGlu Thr Tyr Phe Ser Thr Ala Asp Ser Ile Phe 515 520 525 aat acc gtg ctggcc atg att agg act cga att cag atc cca gct gct 1633 Asn Thr Val Leu AlaMet Ile Arg Thr Arg Ile Gln Ile Pro Ala Ala 530 535 540 ccc atc cgg gaagag gta gca gct gag atc ccc cat cgt tca ggc aag 1681 Pro Ile Arg Glu GluVal Ala Ala Glu Ile Pro His Arg Ser Gly Lys 545 550 555 gca gct ggt actgac ccc aac atg gct gac aat ggg gac ctg gac ctg 1729 Ala Ala Gly Thr AspPro Asn Met Ala Asp Asn Gly Asp Leu Asp Leu 560 565 570 575 ggg ccg gtagag aca gag ccg ccc tat gcc tca cct aaa tac ctc cgt 1777 Gly Pro Val GluThr Glu Pro Pro Tyr Ala Ser Pro Lys Tyr Leu Arg 580 585 590 aat ttc actctg act gtc act gac tgt tac cgc agc tgg aac tcc gca 1825 Asn Phe Thr LeuThr Val Thr Asp Cys Tyr Arg Ser Trp Asn Ser Ala 595 600 605 ccc gga cctttc cat ctt ttt cca cac aca ccc ttt gac cct gtg ctg 1873 Pro Gly Pro PheHis Leu Phe Pro His Thr Pro Phe Asp Pro Val Leu 610 615 620 ccc tct gaggcc aaa ttc ctg ggc tca ggg act gga ttt cgg ccc atc 1921 Pro Ser Glu AlaLys Phe Leu Gly Ser Gly Thr Gly Phe Arg Pro Ile 625 630 635 ggt ggt ggggct ggg ggc tct ggc aag gag ttc cag gca gcg ctt gga 1969 Gly Gly Gly AlaGly Gly Ser Gly Lys Glu Phe Gln Ala Ala Leu Gly 640 645 650 655 ggc aatgtc cag cgg gag cag ttc aca gtt gtg atg ctg acc tac gag 2017 Gly Asn ValGln Arg Glu Gln Phe Thr Val Val Met Leu Thr Tyr Glu 660 665 670 cgg gaggaa gtg ctc atg aac tcc ctg gag agg ctc aat ggc ctc ccc 2065 Arg Glu GluVal Leu Met Asn Ser Leu Glu Arg Leu Asn Gly Leu Pro 675 680 685 tac ctgaac aag gta gtg gtg gtg tgg aac tct ccc aag ctg ccc tcg 2113 Tyr Leu AsnLys Val Val Val Val Trp Asn Ser Pro Lys Leu Pro Ser 690 695 700 gag gacctt ttg tgg cca gac att ggt gtc ccc atc atg gtt gtc cgt 2161 Glu Asp LeuLeu Trp Pro Asp Ile Gly Val Pro Ile Met Val Val Arg 705 710 715 act gagaag aac agt ttg aac aat cgg ttc ttg ccc tgg aat gag ata 2209 Thr Glu LysAsn Ser Leu Asn Asn Arg Phe Leu Pro Trp Asn Glu Ile 720 725 730 735 gagaca gag gca ata ttg tcc atc gat gac gat gcc cac ctc cgc cat 2257 Glu ThrGlu Ala Ile Leu Ser Ile Asp Asp Asp Ala His Leu Arg His 740 745 750 gatgaa atc atg ttc ggg ttt cgg gtg tgg aga gag gcg cgt gat cgc 2305 Asp GluIle Met Phe Gly Phe Arg Val Trp Arg Glu Ala Arg Asp Arg 755 760 765 attgtg ggg ttc cct ggc cgg tac cat gcg tgg gac atc cct cac cag 2353 Ile ValGly Phe Pro Gly Arg Tyr His Ala Trp Asp Ile Pro His Gln 770 775 780 tcctgg ctc tac aac tcc aac tac tcc tgt gag ctg tcc atg gtg ctg 2401 Ser TrpLeu Tyr Asn Ser Asn Tyr Ser Cys Glu Leu Ser Met Val Leu 785 790 795 acgggt gct gcc ttc ttt cac aag tat tac gcc tac ctg tat tct tat 2449 Thr GlyAla Ala Phe Phe His Lys Tyr Tyr Ala Tyr Leu Tyr Ser Tyr 800 805 810 815gtg atg ccc cag gcc atc cga gac atg gtg gat gag tat atc aac tgt 2497 ValMet Pro Gln Ala Ile Arg Asp Met Val Asp Glu Tyr Ile Asn Cys 820 825 830gag gat atc gcc atg aac ttc ctt gtc tcc cac atc aca cgg aag ccc 2545 GluAsp Ile Ala Met Asn Phe Leu Val Ser His Ile Thr Arg Lys Pro 835 840 845ccc atc aag gtg aca tcg agg tgg act ttt cga tgc ccg ggg tgc cct 2593 ProIle Lys Val Thr Ser Arg Trp Thr Phe Arg Cys Pro Gly Cys Pro 850 855 860cag gcc ctg tcc cac gat gac tct cac ttt cat gag cgg cac aag tgt 2641 GlnAla Leu Ser His Asp Asp Ser His Phe His Glu Arg His Lys Cys 865 870 875atc aac ttt ttt gtc aag gtg tac ggc tat atg cct ctc ctg tac aca 2689 IleAsn Phe Phe Val Lys Val Tyr Gly Tyr Met Pro Leu Leu Tyr Thr 880 885 890895 cag ttt agg gtg gac tct gtg ctc ttc aag acc cgc ctg ccc cat gac 2737Gln Phe Arg Val Asp Ser Val Leu Phe Lys Thr Arg Leu Pro His Asp 900 905910 aag acc aag tgc ttc aag ttc atc tagggccttg ccagttctga ggagaagaca2791 Lys Thr Lys Cys Phe Lys Phe Ile 915 gtgagcagag tgaggggagtcacccccaag gttcccaagg tgttgaaggt ccttggggac 2851 atcgtgggca gggcccaggccctttgcttg gagaagagca gggagagtag aaagggatgg 2911 ctgtctttat tttgaagtcagccgcactgg gcctggaatc ctggtcagca gactcagggc 2971 accgactaat ggcgaacactgaggactgtt catgagcccg ggacagctgg ttcccggttt 3031 ttaaattcag aacagcatttactatttaaa gagagagttt cacatctgcc atccaaggct 3091 tatttatatg tgcgtatatgtacacacata tgtgtatata catgtatatg cacgcacaca 3151 cacacacaca cacacacacacacacacaca cacacacagc ggccgcg 3198 4 919 PRT Rattus norvegicus 4 Met ThrGly Tyr Thr Met Leu Arg Asn Gly Gly Val Gly Asn Gly Gly 1 5 10 15 GlnThr Cys Met Leu Arg Trp Ser Asn Arg Ile Arg Leu Thr Trp Leu 20 25 30 SerPhe Thr Leu Phe Ile Ile Leu Val Phe Phe Pro Leu Ile Ala His 35 40 45 TyrTyr Leu Thr Thr Leu Asp Glu Ala Asp Glu Ala Gly Lys Arg Ile 50 55 60 PheGly Pro Arg Ala Gly Asn Glu Leu Cys Glu Val Lys His Val Leu 65 70 75 80Asp Leu Cys Arg Ile Arg Glu Ser Val Ser Glu Glu Leu Leu Gln Leu 85 90 95Glu Ala Lys Arg Gln Glu Leu Asn Ser Glu Ile Ala Lys Leu Asn Leu 100 105110 Lys Ile Glu Ala Cys Lys Lys Ser Ile Glu Asn Ala Lys Gln Asp Leu 115120 125 Leu Gln Leu Lys Asn Val Ile Ser Gln Thr Glu His Ser Tyr Lys Glu130 135 140 Leu Met Ala Gln Asn Gln Pro Lys Leu Ser Leu Pro Ile Arg LeuLeu 145 150 155 160 Pro Glu Lys Asp Asp Ala Gly Leu Pro Pro Pro Lys ValThr Arg Gly 165 170 175 Cys Arg Leu His Asn Cys Phe Asp Tyr Ser Arg CysPro Leu Thr Ser 180 185 190 Gly Phe Pro Val Phe Val Tyr Asp Ser Asp GlnPhe Ala Phe Gly Ser 195 200 205 Tyr Leu Asp Pro Leu Val Lys Gln Ala PheGln Ala Thr Val Arg Ala 210 215 220 Asn Val Tyr Val Thr Glu Asn Ala AlaIle Ala Cys Leu Tyr Val Val 225 230 235 240 Leu Val Gly Glu Ile Gln GluPro Ala Val Leu Gln Pro Ala Asp Leu 245 250 255 Glu Lys Gln Leu His SerLeu Pro His Trp Arg Thr Asp Gly His Asn 260 265 270 His Val Ile Ile AsnLeu Ser Arg Lys Ser Asp Thr Gln Asn Leu Leu 275 280 285 Tyr Asn Val SerThr Gly Arg Ala Met Val Ala Gln Ser Thr Phe Tyr 290 295 300 Ala Ala GlnTyr Arg Ala Gly Phe Asp Leu Val Val Ser Pro Leu Val 305 310 315 320 HisAla Met Ser Glu Pro Asn Phe Met Glu Ile Pro Pro Gln Val Pro 325 330 335Val Lys Arg Lys Tyr Leu Phe Thr Phe Gln Gly Glu Lys Ile Glu Ser 340 345350 Leu Arg Ser Ser Leu Gln Glu Ala Arg Ser Phe Glu Glu Glu Met Glu 355360 365 Gly Asp Pro Pro Ala Asp Tyr Asp Asp Arg Ile Ile Ala Thr Leu Lys370 375 380 Ala Val Gln Asp Ser Lys Leu Asp Gln Val Leu Val Glu Phe ThrCys 385 390 395 400 Lys Asn Gln Pro Lys Pro Ser Leu Pro Thr Glu Trp AlaLeu Cys Gly 405 410 415 Glu Arg Glu Asp Arg Leu Glu Leu Leu Lys Leu SerThr Phe Ala Leu 420 425 430 Ile Ile Thr Pro Gly Asp Pro Ser Leu Leu IleSer Ser Gly Cys Ala 435 440 445 Thr Arg Leu Phe Glu Ala Leu Glu Val GlyAla Val Pro Val Val Leu 450 455 460 Gly Glu Gln Val Gln Leu Pro Tyr HisAsp Met Leu Gln Trp Asn Glu 465 470 475 480 Ala Ala Leu Val Val Pro LysPro Arg Val Thr Glu Val His Phe Leu 485 490 495 Leu Arg Ser Leu Ser AspSer Asp Leu Leu Ala Met Arg Arg Gln Gly 500 505 510 Arg Phe Leu Trp GluThr Tyr Phe Ser Thr Ala Asp Ser Ile Phe Asn 515 520 525 Thr Val Leu AlaMet Ile Arg Thr Arg Ile Gln Ile Pro Ala Ala Pro 530 535 540 Ile Arg GluGlu Val Ala Ala Glu Ile Pro His Arg Ser Gly Lys Ala 545 550 555 560 AlaGly Thr Asp Pro Asn Met Ala Asp Asn Gly Asp Leu Asp Leu Gly 565 570 575Pro Val Glu Thr Glu Pro Pro Tyr Ala Ser Pro Lys Tyr Leu Arg Asn 580 585590 Phe Thr Leu Thr Val Thr Asp Cys Tyr Arg Ser Trp Asn Ser Ala Pro 595600 605 Gly Pro Phe His Leu Phe Pro His Thr Pro Phe Asp Pro Val Leu Pro610 615 620 Ser Glu Ala Lys Phe Leu Gly Ser Gly Thr Gly Phe Arg Pro IleGly 625 630 635 640 Gly Gly Ala Gly Gly Ser Gly Lys Glu Phe Gln Ala AlaLeu Gly Gly 645 650 655 Asn Val Gln Arg Glu Gln Phe Thr Val Val Met LeuThr Tyr Glu Arg 660 665 670 Glu Glu Val Leu Met Asn Ser Leu Glu Arg LeuAsn Gly Leu Pro Tyr 675 680 685 Leu Asn Lys Val Val Val Val Trp Asn SerPro Lys Leu Pro Ser Glu 690 695 700 Asp Leu Leu Trp Pro Asp Ile Gly ValPro Ile Met Val Val Arg Thr 705 710 715 720 Glu Lys Asn Ser Leu Asn AsnArg Phe Leu Pro Trp Asn Glu Ile Glu 725 730 735 Thr Glu Ala Ile Leu SerIle Asp Asp Asp Ala His Leu Arg His Asp 740 745 750 Glu Ile Met Phe GlyPhe Arg Val Trp Arg Glu Ala Arg Asp Arg Ile 755 760 765 Val Gly Phe ProGly Arg Tyr His Ala Trp Asp Ile Pro His Gln Ser 770 775 780 Trp Leu TyrAsn Ser Asn Tyr Ser Cys Glu Leu Ser Met Val Leu Thr 785 790 795 800 GlyAla Ala Phe Phe His Lys Tyr Tyr Ala Tyr Leu Tyr Ser Tyr Val 805 810 815Met Pro Gln Ala Ile Arg Asp Met Val Asp Glu Tyr Ile Asn Cys Glu 820 825830 Asp Ile Ala Met Asn Phe Leu Val Ser His Ile Thr Arg Lys Pro Pro 835840 845 Ile Lys Val Thr Ser Arg Trp Thr Phe Arg Cys Pro Gly Cys Pro Gln850 855 860 Ala Leu Ser His Asp Asp Ser His Phe His Glu Arg His Lys CysIle 865 870 875 880 Asn Phe Phe Val Lys Val Tyr Gly Tyr Met Pro Leu LeuTyr Thr Gln 885 890 895 Phe Arg Val Asp Ser Val Leu Phe Lys Thr Arg LeuPro His Asp Lys 900 905 910 Thr Lys Cys Phe Lys Phe Ile 915 5 919 PRTHomo sapiens 5 Met Thr Gly Tyr Thr Met Leu Arg Asn Gly Gly Ala Gly AsnGly Gly 1 5 10 15 Gln Thr Cys Met Leu Arg Trp Ser Asn Arg Ile Arg LeuThr Trp Leu 20 25 30 Ser Phe Thr Leu Phe Val Ile Leu Val Phe Phe Pro LeuIle Ala His 35 40 45 Tyr Tyr Leu Thr Thr Leu Asp Glu Ala Asp Glu Ala GlyLys Arg Ile 50 55 60 Phe Gly Pro Arg Val Gly Asn Glu Leu Cys Glu Val LysHis Val Leu 65 70 75 80 Asp Leu Cys Arg Ile Arg Glu Ser Val Ser Glu GluLeu Leu Gln Leu 85 90 95 Glu Ala Lys Arg Gln Glu Leu Asn Ser Glu Ile AlaLys Leu Asn Leu 100 105 110 Lys Ile Glu Ala Cys Lys Lys Ser Ile Glu AsnAla Lys Gln Asp Leu 115 120 125 Leu Gln Leu Lys Asn Val Ile Ser Gln ThrGlu His Ser Tyr Lys Glu 130 135 140 Leu Met Ala Gln Asn Gln Pro Lys LeuSer Leu Pro Ile Arg Leu Leu 145 150 155 160 Pro Glu Lys Asp Asp Ala GlyLeu Pro Pro Pro Lys Ala Thr Arg Gly 165 170 175 Cys Arg Leu His Asn CysPhe Asp Tyr Ser Arg Cys Pro Leu Thr Ser 180 185 190 Gly Phe Pro Val TyrVal Tyr Asp Ser Asp Gln Phe Val Phe Gly Ser 195 200 205 Tyr Leu Asp ProLeu Val Lys Gln Ala Phe Gln Ala Thr Ala Arg Ala 210 215 220 Asn Val TyrVal Thr Glu Asn Ala Asp Ile Ala Cys Leu Tyr Val Ile 225 230 235 240 LeuVal Gly Glu Met Gln Glu Pro Val Val Leu Arg Pro Ala Glu Leu 245 250 255Glu Lys Gln Leu Tyr Ser Leu Pro His Trp Arg Thr Asp Gly His Asn 260 265270 His Val Ile Ile Asn Leu Ser Arg Lys Ser Asp Thr Gln Asn Leu Leu 275280 285 Tyr Asn Val Ser Thr Gly Arg Ala Met Val Ala Gln Ser Thr Phe Tyr290 295 300 Thr Val Gln Tyr Arg Pro Gly Phe Asp Leu Val Val Ser Pro LeuVal 305 310 315 320 His Ala Met Ser Glu Pro Asn Phe Met Glu Ile Pro ProGln Val Pro 325 330 335 Val Lys Arg Lys Tyr Leu Phe Thr Phe Gln Gly GluLys Ile Glu Ser 340 345 350 Leu Arg Ser Ser Leu Gln Glu Ala Arg Ser PheGlu Glu Glu Met Glu 355 360 365 Gly Asp Pro Pro Ala Asp Tyr Asp Asp ArgIle Ile Ala Thr Leu Lys 370 375 380 Ala Val Gln Asp Ser Lys Leu Asp GlnVal Leu Val Glu Phe Thr Cys 385 390 395 400 Lys Asn Gln Pro Lys Pro SerLeu Pro Thr Glu Trp Ala Leu Cys Gly 405 410 415 Glu Arg Glu Asp Arg LeuGlu Leu Leu Lys Leu Ser Thr Phe Ala Leu 420 425 430 Ile Ile Thr Pro GlyAsp Pro Arg Leu Val Ile Ser Ser Gly Cys Ala 435 440 445 Thr Arg Leu PheGlu Ala Leu Glu Val Gly Ala Val Pro Val Val Leu 450 455 460 Gly Glu GlnVal Gln Leu Pro Tyr Gln Asp Met Leu Gln Trp Asn Glu 465 470 475 480 AlaAla Leu Val Val Pro Lys Pro Arg Val Thr Glu Val His Phe Leu 485 490 495Leu Arg Ser Leu Ser Asp Ser Asp Leu Leu Ala Met Arg Arg Gln Gly 500 505510 Arg Phe Leu Trp Glu Thr Tyr Phe Ser Thr Ala Asp Ser Ile Phe Asn 515520 525 Thr Val Leu Ala Met Ile Arg Thr Arg Ile Gln Ile Pro Ala Ala Pro530 535 540 Ile Arg Glu Glu Ala Ala Ala Glu Ile Pro His Arg Ser Gly LysAla 545 550 555 560 Ala Gly Thr Asp Pro Asn Met Ala Asp Asn Gly Asp LeuAsp Leu Gly 565 570 575 Pro Val Glu Thr Glu Pro Pro Tyr Ala Ser Pro ArgTyr Leu Arg Asn 580 585 590 Phe Thr Leu Thr Val Thr Asp Phe Tyr Arg SerTrp Asn Cys Ala Pro 595 600 605 Gly Pro Phe His Leu Phe Pro His Thr ProPhe Asp Pro Val Leu Pro 610 615 620 Ser Glu Ala Lys Phe Leu Gly Ser GlyThr Gly Phe Arg Pro Ile Gly 625 630 635 640 Gly Gly Ala Gly Gly Ser GlyLys Glu Phe Gln Ala Ala Leu Gly Gly 645 650 655 Asn Val Pro Arg Glu GlnPhe Thr Val Val Met Leu Thr Tyr Glu Arg 660 665 670 Glu Glu Val Leu MetAsn Ser Leu Glu Arg Leu Asn Gly Leu Pro Tyr 675 680 685 Leu Asn Lys ValVal Val Val Trp Asn Ser Pro Lys Leu Pro Ser Glu 690 695 700 Asp Leu LeuTrp Pro Asp Ile Gly Val Pro Ile Met Val Val Arg Thr 705 710 715 720 GluLys Asn Ser Leu Asn Asn Arg Phe Leu Pro Trp Asn Glu Ile Glu 725 730 735Thr Glu Ala Ile Leu Ser Ile Asp Asp Asp Ala His Leu Arg His Asp 740 745750 Glu Ile Met Phe Gly Phe Arg Val Trp Arg Glu Ala Arg Asp Arg Ile 755760 765 Val Gly Phe Pro Gly Arg Tyr His Ala Trp Asp Ile Pro His Gln Ser770 775 780 Trp Leu Tyr Asn Ser Asn Tyr Ser Cys Glu Leu Ser Met Val LeuThr 785 790 795 800 Gly Ala Ala Phe Phe His Lys Tyr Tyr Ala Tyr Leu TyrSer Tyr Val 805 810 815 Met Pro Gln Ala Ile Arg Asp Met Val Asp Glu TyrIle Asn Cys Glu 820 825 830 Asp Ile Ala Met Asn Phe Leu Val Ser His IleThr Arg Lys Pro Pro 835 840 845 Ile Lys Val Thr Ser Arg Trp Thr Phe ArgCys Pro Gly Cys Pro Gln 850 855 860 Ala Leu Ser His Asp Asp Ser His PheHis Glu Arg His Lys Cys Ile 865 870 875 880 Asn Phe Phe Val Lys Val TyrGly Tyr Met Pro Leu Leu Tyr Thr Gln 885 890 895 Phe Arg Val Asp Ser ValLeu Phe Lys Thr Arg Leu Pro His Asp Lys 900 905 910 Thr Lys Cys Phe LysPhe Ile 915 6 718 PRT Homo sapiens 6 Met Cys Ala Ser Val Lys Tyr Asn IleArg Gly Pro Ala Leu Ile Pro 1 5 10 15 Arg Met Lys Thr Lys His Arg IleTyr Tyr Ile Thr Leu Phe Ser Ile 20 25 30 Val Leu Leu Gly Leu Ile Ala ThrGly Met Phe Gln Phe Trp Pro His 35 40 45 Ser Ile Glu Ser Ser Asn Asp TrpAsn Val Glu Lys Arg Ser Ile Arg 50 55 60 Asp Val Pro Val Val Arg Leu ProAla Asp Ser Pro Ile Pro Glu Arg 65 70 75 80 Gly Asp Leu Ser Cys Arg MetHis Thr Cys Phe Asp Val Tyr Arg Cys 85 90 95 Gly Phe Asn Pro Lys Asn LysIle Lys Val Tyr Ile Tyr Ala Leu Lys 100 105 110 Lys Tyr Val Asp Asp PheGly Val Ser Val Ser Asn Thr Ile Ser Arg 115 120 125 Glu Tyr Asn Glu LeuLeu Met Ala Ile Ser Asp Ser Asp Tyr Tyr Thr 130 135 140 Asp Asp Ile AsnArg Ala Cys Leu Phe Val Pro Ser Ile Asp Val Leu 145 150 155 160 Asn GlnAsn Thr Leu Arg Ile Lys Glu Thr Ala Gln Ala Met Ala Gln 165 170 175 LeuSer Arg Trp Asp Arg Gly Thr Asn His Leu Leu Phe Asn Met Leu 180 185 190Pro Gly Gly Pro Pro Asp Tyr Asn Thr Ala Leu Asp Val Pro Arg Asp 195 200205 Arg Ala Leu Leu Ala Gly Gly Gly Phe Ser Thr Trp Thr Tyr Arg Gln 210215 220 Gly Tyr Asp Val Ser Ile Pro Val Tyr Ser Pro Leu Ser Ala Glu Val225 230 235 240 Asp Leu Pro Glu Lys Gly Pro Gly Pro Arg Gln Tyr Phe LeuLeu Ser 245 250 255 Ser Gln Val Gly Leu His Pro Glu Tyr Arg Glu Asp LeuGlu Ala Leu 260 265 270 Gln Val Lys His Gly Glu Ser Val Leu Val Leu AspLys Cys Thr Asn 275 280 285 Leu Ser Glu Gly Val Leu Ser Val Arg Lys ArgCys His Lys His Gln 290 295 300 Val Phe Asp Tyr Pro Gln Val Leu Gln GluAla Thr Phe Cys Val Val 305 310 315 320 Leu Arg Gly Ala Arg Leu Gly GlnAla Val Leu Ser Asp Val Leu Gln 325 330 335 Ala Gly Cys Val Pro Val ValIle Ala Asp Ser Tyr Ile Leu Pro Phe 340 345 350 Ser Glu Val Leu Asp TrpLys Arg Ala Ser Val Val Val Pro Glu Glu 355 360 365 Lys Met Ser Asp ValTyr Ser Ile Leu Gln Ser Ile Pro Gln Arg Gln 370 375 380 Ile Glu Glu MetGln Arg Gln Ala Arg Trp Phe Trp Glu Ala Tyr Phe 385 390 395 400 Gln SerIle Lys Ala Ile Ala Leu Ala Thr Leu Gln Ile Ile Asn Asp 405 410 415 ArgIle Tyr Pro Tyr Ala Ala Ile Ser Tyr Glu Glu Trp Asn Asp Pro 420 425 430Pro Ala Val Lys Trp Gly Ser Val Ser Asn Pro Leu Phe Leu Pro Leu 435 440445 Ile Pro Pro Gln Ser Gln Gly Phe Thr Ala Ile Val Leu Thr Tyr Asp 450455 460 Arg Val Glu Ser Leu Phe Arg Val Ile Thr Glu Val Ser Lys Val Pro465 470 475 480 Ser Leu Ser Lys Leu Leu Val Val Trp Asn Asn Gln Asn LysAsn Pro 485 490 495 Pro Glu Asp Ser Leu Trp Pro Lys Ile Arg Val Pro LeuLys Val Val 500 505 510 Arg Thr Ala Glu Asn Lys Leu Ser Asn Arg Phe PhePro Tyr Asp Glu 515 520 525 Ile Glu Thr Glu Ala Val Leu Ala Ile Asp AspAsp Ile Ile Met Leu 530 535 540 Thr Ser Asp Glu Leu Gln Phe Gly Tyr GluVal Trp Arg Glu Phe Pro 545 550 555 560 Asp Arg Leu Val Gly Tyr Pro GlyArg Leu His Leu Trp Asp His Glu 565 570 575 Met Asn Lys Trp Lys Tyr GluSer Glu Trp Thr Asn Glu Val Ser Met 580 585 590 Val Leu Thr Gly Ala AlaPhe Tyr His Lys Tyr Phe Asn Tyr Leu Tyr 595 600 605 Thr Tyr Lys Met ProGly Asp Ile Lys Asn Trp Val Asp Ala His Met 610 615 620 Asn Cys Glu AspIle Ala Met Asn Phe Leu Val Ala Asn Val Thr Gly 625 630 635 640 Lys AlaVal Ile Lys Val Thr Pro Arg Lys Lys Phe Lys Cys Pro Glu 645 650 655 CysThr Ala Ile Asp Gly Leu Ser Leu Asp Gln Thr His Met Val Glu 660 665 670Arg Ser Glu Cys Ile Asn Lys Phe Ala Ser Val Phe Gly Thr Met Pro 675 680685 Leu Lys Val Val Glu His Arg Ala Asp Pro Val Leu Tyr Lys Asp Asp 690695 700 Phe Pro Glu Lys Leu Lys Ser Phe Pro Asn Ile Gly Ser Leu 705 710715 7 746 PRT Homo sapiens 7 Met Gln Ala Lys Lys Arg Tyr Phe Ile Leu LeuSer Ala Gly Ser Cys 1 5 10 15 Leu Ala Leu Leu Phe Tyr Phe Gly Gly LeuGln Phe Arg Ala Ser Arg 20 25 30 Ser His Ser Arg Arg Glu Glu His Ser GlyArg Asn Gly Leu His His 35 40 45 Pro Ser Pro Asp His Phe Trp Pro Arg PhePro Glu Pro Leu Arg Pro 50 55 60 Phe Val Pro Trp Asp Gln Leu Glu Asn GluAsp Ser Ser Val His Ile 65 70 75 80 Ser Pro Arg Gln Lys Arg Asp Ala AsnSer Ser Ile Tyr Lys Gly Lys 85 90 95 Lys Cys Arg Met Glu Ser Cys Phe AspPhe Thr Leu Cys Lys Lys Asn 100 105 110 Gly Phe Lys Val Tyr Val Tyr ProGln Gln Lys Gly Glu Lys Ile Ala 115 120 125 Glu Ser Tyr Gln Asn Ile LeuAla Ala Ile Glu Gly Ser Arg Phe Tyr 130 135 140 Thr Ser Asp Pro Ser GlnAla Cys Leu Phe Val Leu Ser Leu Asp Thr 145 150 155 160 Leu Asp Arg AspGln Leu Ser Pro Gln Tyr Val His Asn Leu Arg Ser 165 170 175 Lys Val GlnSer Leu His Leu Trp Asn Asn Gly Arg Asn His Leu Ile 180 185 190 Phe AsnLeu Tyr Ser Gly Thr Trp Pro Asp Tyr Thr Glu Asp Val Gly 195 200 205 PheAsp Ile Gly Gln Ala Met Leu Ala Lys Ala Ser Ile Ser Thr Glu 210 215 220Asn Phe Arg Pro Asn Phe Asp Val Ser Ile Pro Leu Phe Ser Lys Asp 225 230235 240 His Pro Arg Thr Gly Gly Glu Arg Gly Phe Leu Lys Phe Asn Thr Ile245 250 255 Pro Pro Leu Arg Lys Tyr Met Leu Val Phe Lys Gly Lys Arg TyrLeu 260 265 270 Thr Gly Ile Gly Ser Asp Thr Arg Asn Ala Leu Tyr His ValHis Asn 275 280 285 Gly Glu Asp Val Val Leu Leu Thr Thr Cys Lys His GlyLys Asp Trp 290 295 300 Gln Lys His Lys Asp Ser Arg Cys Asp Arg Asp AsnThr Glu Tyr Glu 305 310 315 320 Lys Tyr Asp Tyr Arg Glu Met Leu His AsnAla Thr Phe Cys Leu Val 325 330 335 Pro Arg Gly Arg Arg Leu Gly Ser PheArg Phe Leu Glu Ala Leu Gln 340 345 350 Ala Ala Cys Val Pro Val Met LeuSer Asn Gly Trp Glu Leu Pro Phe 355 360 365 Ser Glu Val Ile Asn Trp AsnGln Ala Ala Val Ile Gly Asp Glu Arg 370 375 380 Leu Leu Leu Gln Ile ProSer Thr Ile Arg Ser Ile His Gln Asp Lys 385 390 395 400 Ile Leu Ala LeuArg Gln Gln Thr Gln Phe Leu Trp Glu Ala Tyr Phe 405 410 415 Ser Ser ValGlu Lys Ile Val Leu Thr Thr Leu Glu Ile Ile Gln Asp 420 425 430 Arg IlePhe Lys His Ile Ser Arg Asn Ser Leu Ile Trp Asn Lys His 435 440 445 ProGly Gly Leu Phe Val Leu Pro Gln Tyr Ser Ser Tyr Leu Gly Asp 450 455 460Phe Pro Tyr Tyr Tyr Ala Asn Leu Gly Leu Lys Pro Pro Ser Lys Phe 465 470475 480 Thr Ala Val Ile His Ala Val Thr Pro Leu Val Ser Gln Ser Gln Pro485 490 495 Val Leu Lys Leu Leu Val Ala Ala Ala Lys Ser Gln Tyr Cys AlaGln 500 505 510 Ile Ile Val Leu Trp Asn Cys Asp Lys Pro Leu Pro Ala LysHis Arg 515 520 525 Trp Pro Ala Thr Ala Val Pro Val Val Val Ile Glu GlyGlu Ser Lys 530 535 540 Val Met Ser Ser Arg Phe Leu Pro Tyr Asp Asn IleIle Thr Asp Ala 545 550 555 560 Val Leu Ser Leu Asp Glu Asp Thr Val LeuSer Thr Thr Glu Val Asp 565 570 575 Phe Ala Phe Thr Val Trp Gln Ser PhePro Glu Arg Ile Val Gly Tyr 580 585 590 Pro Ala Arg Ser His Phe Trp AspAsn Ser Lys Glu Arg Trp Gly Tyr 595 600 605 Thr Ser Lys Trp Thr Asn AspTyr Ser Met Val Leu Thr Gly Ala Ala 610 615 620 Ile Tyr His Lys Tyr TyrHis Tyr Leu Tyr Ser His Tyr Leu Pro Ala 625 630 635 640 Ser Leu Lys AsnMet Val Asp Gln Leu Ala Asn Cys Glu Asp Ile Leu 645 650 655 Met Asn PheLeu Val Ser Ala Val Thr Lys Leu Pro Pro Ile Lys Val 660 665 670 Thr GlnLys Lys Gln Tyr Lys Glu Thr Met Met Gly Gln Thr Ser Arg 675 680 685 AlaSer Arg Trp Ala Asp Pro Asp His Phe Ala Gln Arg Gln Ser Cys 690 695 700Met Asn Thr Phe Ala Ser Trp Phe Gly Tyr Met Pro Leu Ile His Ser 705 710715 720 Gln Met Arg Leu Asp Pro Val Leu Phe Lys Asp Gln Val Ser Ile Leu725 730 735 Arg Lys Lys Tyr Arg Asp Ile Glu Arg Leu 740 745 8 676 PRTHomo sapiens 8 Met Gln Ser Trp Arg Arg Arg Lys Ser Leu Trp Leu Ala LeuSer Ala 1 5 10 15 Ser Trp Leu Leu Leu Val Leu Leu Gly Gly Phe Ser LeuLeu Arg Leu 20 25 30 Ala Leu Pro Pro Arg Pro Arg Pro Gly Ala Ser Gln GlyTrp Pro Arg 35 40 45 Trp Leu Asp Ala Glu Leu Leu Gln Ser Phe Ser Gln ProGly Glu Leu 50 55 60 Pro Glu Asp Ala Val Ser Pro Pro Gln Ala Pro His GlyGly Ser Cys 65 70 75 80 Asn Trp Glu Ser Cys Phe Asp Thr Ser Lys Cys ArgGly Asp Gly Leu 85 90 95 Lys Val Phe Val Tyr Pro Ala Val Gly Thr Ile SerGlu Thr His Arg 100 105 110 Arg Ile Leu Ala Ser Ile Glu Gly Ser Arg PheTyr Thr Phe Ser Pro 115 120 125 Ala Gly Ala Cys Leu Leu Leu Leu Leu SerLeu Asp Ala Gln Thr Gly 130 135 140 Glu Cys Ser Ser Met Pro Leu Gln TrpAsn Arg Gly Arg Asn His Leu 145 150 155 160 Val Leu Arg Leu His Pro AlaPro Cys Pro Arg Thr Phe Gln Leu Gly 165 170 175 Gln Ala Met Val Ala GluAla Ser Pro Thr Val Asp Ser Phe Arg Pro 180 185 190 Gly Phe Asp Val AlaLeu Pro Phe Leu Pro Glu Ala His Pro Leu Arg 195 200 205 Gly Gly Ala ProGly Gln Leu Arg Gln His Ser Pro Gln Pro Gly Val 210 215 220 Ala Leu LeuAla Leu Glu Glu Glu Arg Gly Gly Trp Arg Thr Ala Asp 225 230 235 240 ThrGly Ser Ser Ala Cys Pro Trp Asp Gly Arg Cys Glu Gln Asp Pro 245 250 255Gly Pro Gly Gln Thr Gln Arg Gln Glu Thr Leu Pro Asn Ala Thr Phe 260 265270 Cys Leu Ile Ser Gly His Arg Pro Glu Ala Ala Ser Arg Phe Leu Gln 275280 285 Ala Leu Gln Ala Gly Cys Ile Pro Val Leu Leu Ser Pro Arg Trp Glu290 295 300 Leu Pro Phe Ser Glu Val Ile Asp Trp Thr Lys Ala Ala Ile ValAla 305 310 315 320 Asp Glu Arg Leu Pro Leu Gln Val Leu Ala Ala Leu GlnGlu Met Ser 325 330 335 Pro Ala Arg Val Leu Ala Leu Arg Gln Gln Thr GlnPhe Leu Trp Asp 340 345 350 Ala Tyr Phe Ser Ser Val Glu Lys Val Ile HisThr Thr Leu Glu Val 355 360 365 Ile Gln Asp Arg Ile Phe Gly Thr Ser AlaAsn Pro Ser Leu Leu Trp 370 375 380 Asn Ser Pro Pro Gly Ala Leu Leu AlaLeu Ser Thr Phe Ser Thr Ser 385 390 395 400 Pro Gln Asp Phe Pro Phe TyrTyr Leu Gln Gln Gly Ser Arg Pro Glu 405 410 415 Gly Arg Phe Ser Ala LeuIle Trp Val Gly Pro Pro Gly Gln Pro Pro 420 425 430 Leu Lys Leu Ile GlnAla Val Ala Gly Ser Gln His Cys Ala Gln Ile 435 440 445 Leu Val Leu TrpSer Asn Glu Arg Pro Leu Pro Ser Arg Trp Pro Glu 450 455 460 Thr Ala ValPro Leu Thr Val Ile Asp Gly His Arg Lys Val Ser Asp 465 470 475 480 ArgPhe Tyr Pro Tyr Ser Thr Ile Arg Thr Asp Ala Ile Leu Ser Leu 485 490 495Asp Ala Arg Ser Ser Leu Ser Thr Ser Glu Val Asp Phe Ala Phe Leu 500 505510 Val Trp Gln Ser Phe Pro Glu Arg Met Val Gly Phe Leu Thr Ser Ser 515520 525 His Phe Trp Asp Glu Ala His Gly Gly Trp Gly Tyr Thr Ala Glu Arg530 535 540 Thr Asn Glu Phe Ser Met Val Leu Thr Thr Ala Ala Phe Tyr HisArg 545 550 555 560 Tyr Tyr His Thr Leu Phe Thr His Ser Leu Pro Lys AlaLeu Arg Thr 565 570 575 Leu Ala Asp Glu Ala Pro Thr Cys Val Asp Val LeuMet Asn Phe Ile 580 585 590 Val Ala Ala Val Thr Lys Leu Pro Pro Ile LysVal Pro Tyr Gly Lys 595 600 605 Gln Arg Gln Glu Ala Ala Pro Leu Ala ProGly Gly Pro Gly Pro Arg 610 615 620 Pro Lys Pro Pro Ala Pro Ala Pro AspCys Ile Asn Gln Ile Ala Ala 625 630 635 640 Ala Phe Gly His Met Pro LeuLeu Ser Ser Arg Leu Arg Leu Asp Pro 645 650 655 Val Leu Phe Lys Asp ProVal Ser Val Gln Arg Lys Lys Tyr Arg Ser 660 665 670 Leu Glu Lys Pro 6759 330 PRT Homo sapiens 9 Met Arg Cys Cys His Ile Cys Lys Leu Pro Gly ArgVal Met Gly Ile 1 5 10 15 Arg Val Leu Arg Leu Ser Leu Val Val Ile LeuVal Leu Leu Leu Val 20 25 30 Ala Gly Ala Leu Thr Ala Leu Leu Pro Ser ValLys Glu Asp Lys Met 35 40 45 Leu Met Leu Arg Arg Glu Ile Lys Ser Gln GlyLys Ser Thr Met Asp 50 55 60 Ser Phe Thr Leu Ile Met Gln Thr Tyr Asn ArgThr Asp Leu Leu Leu 65 70 75 80 Lys Leu Leu Asn His Tyr Gln Ala Val ProAsn Leu His Lys Val Ile 85 90 95 Val Val Trp Asn Asn Ile Gly Glu Lys AlaPro Asp Glu Leu Trp Asn 100 105 110 Ser Leu Gly Pro His Pro Ile Pro ValIle Phe Lys Gln Gln Thr Ala 115 120 125 Asn Arg Met Arg Asn Arg Leu GlnVal Phe Pro Glu Leu Glu Thr Asn 130 135 140 Ala Val Leu Met Val Asp AspAsp Thr Leu Ile Ser Thr Pro Asp Leu 145 150 155 160 Val Phe Ala Phe SerVal Trp Gln Gln Phe Pro Asp Gln Ile Val Gly 165 170 175 Phe Val Pro ArgLys His Val Ser Thr Ser Ser Gly Ile Tyr Ser Tyr 180 185 190 Gly Ser PheGlu Met Gln Ala Pro Gly Ser Gly Asn Gly Asp Gln Tyr 195 200 205 Ser MetVal Leu Ile Gly Ala Ser Phe Phe Asn Ser Lys Tyr Leu Glu 210 215 220 LeuPhe Gln Arg Gln Pro Ala Ala Val His Ala Leu Ile Asp Asp Thr 225 230 235240 Gln Asn Cys Asp Asp Ile Ala Met Asn Phe Ile Ile Ala Lys His Ile 245250 255 Gly Lys Thr Ser Gly Ile Phe Val Lys Pro Val Asn Met Asp Asn Leu260 265 270 Glu Lys Glu Thr Asn Ser Gly Tyr Ser Gly Met Trp His Arg AlaGlu 275 280 285 His Ala Leu Gln Arg Ser Tyr Cys Ile Asn Lys Leu Val AsnIle Tyr 290 295 300 Asp Ser Met Pro Leu Arg Tyr Ser Asn Ile Met Ile SerGln Phe Gly 305 310 315 320 Phe Pro Tyr Ala Asn Tyr Lys Arg Lys Ile 325330 10 9 PRT Influenza virus 10 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5

1-10. (canceled)
 11. A method of screening for one or more compoundsthat promote or inhibit signal transduction caused by an activation of aprotein, wherein said method comprises the following steps of, (a)contacting Reg protein with a cell expressing said the protein on thecell surface, in the presence of a test sample containing one or morecompounds, (b) detecting a change of the cell in response to stimulationby Reg protein, and (c) selecting the one or more compounds that enhanceor suppress the change of the cell as compared to when detected in theabsence of the test sample; wherein said protein is encoded by DNAselected from the group consisting of: (i) a DNA encoding an amino acidsequence comprising SEQ ID NO:2, (ii) a DNA comprising the codingsequence of SEQ ID NO:1, (iii) a DNA encoding an amino acid sequence inwhich one or more amino acids of SEQ ID NO:2 have been substituted,deleted, inserted and/or added, wherein said protein binds to Regprotein. (iv) a DNA hybridizing to the nucleotide sequence of SEQ IDNO:1, wherein said protein binds to Reg protein, (v) a DNA encoding anamino acid sequence comprising SEQ ID NO:4, (vi) a DNA comprising thecoding region of SEQ ID NO:3, (vii) a DNA encoding an amino acidsequence in which one or more amino acids of SEQ ID NO:4 have beensubstituted, deleted, inserted and/or added, wherein said protein bindsto Reg protein, (viii) a DNA hybridizing to the nucleotide sequence ofSEQ ID NO:3, wherein said protein binds to Reg protein, and (ix) a DNAencoding a partial amino acid sequence of SEQ ID NO:2 or SEQ ID No:4.12. The method according to claim 11, wherein said change of the celldetected comprises a change in cell-proliferating activity orDNA-synthesizing activity of the cell. 13-16. (canceled)